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About This User Guide This user guide is a practical guide to using

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1. SJS SJS lt GF a a a a a a a a a HEE PRPs SYS ILRI TSI LSI SY SY SI SY SY SSS ST SY SSS SS SSSI Chains Se ee ee ses SES TESTES TESTES TESTES TESTES IES IES Waters BIEBER ARE 9 4 Analysis of Superimposed Proteins Binding Sites A detailed analysis of the superimposed binding sites is shown in the analysis table Information is provided on backbone and side chain movements in the protein ligand overlap conserved water molecules etc Relibase User Guide 89 Relibase Home Text Search Sequence Search Smiles Search Sketcher Hitlists Stored Results Help Superposition Analysis retibase 2 1 0 v solvent edb1u65 A_1 y ligand v chains v solvent odb1w4hA_ v igand v solvent odb tyra v ligand v solvent odh1yxo A_1 v iigand v solvent pdbivot _i v ligand v solvent pdbIaih A_1 v chains v solvent Show All Reliview Controller Show in Reliview Automatic Visualiser Updates I Download Superimposed Structures in MOL2 format Current reference chain PDB 1acj Current reference ligand THA_999_pdb1acj_1 Protein RMS C alpha Sidechain Mutations and Ligand Conserved Chain A Movements Movements Insertions Overlap Waters ChangelReteroncesGnall PDB tacl 0 375 None 1 8 None AD 42 2 C n a 0354 1 10 None 33 37 2 c PDB 1ax9 0 362 None None None BASALA 2
2. e crystallographic_data_required true false This flag determines whether the CRYST1 or SCALEn records are required or not e crystl_z_value_required true false Relibase User Guide 161 162 This flag determines whether or not the z value of the CRYST1 record is required or not Most refinement packages do not write out the z value to the PDB files that they produce deposition_date_regquired_in_file true false If the deposition date is not in the PDB file this flag will prompt the user to add a date to the file If this flag is set to false the date will be set to that when the structure was uploaded into Relibase always_use_current_date_as_deposition_date true false This flag determines if today s date if the flag is set to true or the date from the PDB HEADER if the flag is set to false should be used as the deposition date negative_residue_numbers_permitted true false This flag determines whether or not negative residue numbers are permitted synonym_file path to file This flag allows users to specify alternate ligand three letter code synonym files Relative paths are set to SRELIBASE_ROOT so if you had a file called alt_synonyms txt in the SRELIBASE_ROOT processing directory you could specify it using the flag residue_name_synonym_file processing alt_synonyms txt e auto_accept_ligand_templates true false This flag can be used to automatically accept the ligand atom
3. 10 3 Displaying Geometric Objects in the Draw Window A defined object may be displayed by clicking on its name in the Defined Objects list This may be found in the Geometric Parameters dialogue box opened by hitting the Add 3d button 10 4 Deleting a Geometric Object e An object may be deleted by opening the Geometric Parameters dialogue box click on the Add 3d button clicking on the object name in the Defined Objects list and then hitting the Delete button underneath this list 11 Geometric Parameters e Geometric parameters can be defined when drawing a substructure in the drawing window e Histograms of these parameters can then be viewed after the search has been run e Geometric parameters can also be used to set up 3D substructure searches e g a search for a substructure in which a distance has been constrained to a particular range 11 1 Valid Geometric Parameters Valid geometric parameters are e Distances between atoms and or objects the atoms do not need to be bonded to each other e Angles between atoms and or objects the atoms do not need to be bonded to one another e Torsion angles involving atoms and or objects the atoms do not need to be bonded to one another Relibase User Guide 139 11 2 Defining Geometric Parameters Involving Atoms 140 Geometric parameters must be explicitly defined in the Draw window in order to be displayed as a histograms after the search has been run Open up
4. Helices Sheets amp Strands Turns Sheet Properties Strand Properties Kink Properties Position Ignore Strand Position asidue _ Allow Residue Al Other F O R Adjacent Strands Sense v Ignore adjacent strands sense C Allow Anti Para is Allow Parallel inds _ Allow Mixe Strand Length Minimum Length oH Maximum Length 1 000 Reset Relibase User Guide 151 From within the Strand Properties tab it is possible to constrain a search to look at individual strands that may or may not be in a sheet Properties concerning the position of the strand and the sense of the adjacent strand can be defined by deactivating the Ignore Strand Position and Ignore adjacent strands sense check boxes and selecting from the resultant options The strand length can be constrained to contain a user defined number of residues Use the Reset button to return the settings to their original display Kink Properties Helices Sheets amp Strands Turns Sheet Properties Strand Properties Kink Properties Kinks v Ignore strand kinks Reset e Kinks in a sheet can be defined from within the Kink Properties tab A kink in a sheet is similar to a kink in a helix each sheet has a directional vector associated with each strand If this vector changes within a given strand the residue where the directional change occurs is said to be kinked
5. Alternatively if the match_against_pdb_templates option is set to true the exceptions file can be used to list three letter codes that should never be matched for example LIG INH UNK To list such exceptions prefix the three letter code with an exclamation mark for example LIG INH UNK Note that relative paths will be assumed to start from RELIBASE_ROOT so to specify the file SRELIBASE_ROOT processing exceptions txt you would use the line pdb_templates_exceptions_file processing excepitions txt 6 Structure factors and electron densities e Relibase can be used to store structure factors with pre calculated map coefficients for both 2Fo Fc and Fo Fc maps and to display electron density maps In order to store the structure factors MTZ files can be uploaded along with the associated PDB at the data input stage of the data processing e The structure factors need to be in MTZ file format In order for Relibase to parse MTZ files it needs to know the column names of the map coefficient data These typically depend on the software used to generate the MTZ file At the moment Relibase can handle files from the following third party software e Refmac Attp www ccp4 ac uk html refmac5S html column names FWT PHWT DELFWT PHDELWT e autoBUSTER http www globalphasing com column names 2FOFCWT PH2FOFCWT FOFCWT PHFOFCWT e Phenix ttp w
6. Relibase User Guide 193 N ar aromatic nitrogen O 2 carbonyl oxygen 0 3 ether oxygen O co2 carboxylate phosphate oxygen S 2 thiocarbonyl sulphur S 3 thioether sulphur S o sulphoxide sulphur S 02 sulphone sulphur P 3 phosphate phosphorus Halogens standard element symbols e g F Cl Br I Metals standard element symbols e g Zn Fe Ca Buried Atoms An atom is counted as buried if no part of it is solvent accessible see Solvent Accessibility page 195 It does not matter what types of atoms render the atom inaccessible i e whether the atom is buried by protein or ligand atoms or a combination of both Donatable or Polar Hydrogens A donatable or polar hydrogen is any hydrogen that can be donated in a hydrogen bond This includes any oxygen nitrogen or sulphur bound H atom Hydrogen Bonds By default a protein ligand hydrogen bond is present if e it involves a recognised polar hydrogen see Donatable or Polar Hydrogens page 194 and a recognised acceptor atom see Acceptor Atoms page 193 and e the H acceptor distance is less than the sum of van der Waals radii and the donor H acceptor angle is greater than 90 These criteria can be customised when setting up H bond descriptors please refer to the relevant section of the Hermes documentation for further information Intramolecular hydrogen bonds either in the protein or the ligand are not recognised
7. Deselects everything in the sketcher see Selecting Atoms Sec tion 2 7 page 123 Deletes everything that is selected in the sketcher see Deleting Atoms and Bonds Section 2 8 page 125 Copies all selected atoms in the sketcher see Duplicating Sub structures Copy and Paste Section 8 4 page 137 178 Relibase User Guide APPENDIX B Making the Most Out of Visualisation e The ability to easily and conveniently visualise proteins and protein ligand complexes is central to the design of Reliabse Below are some suggestions on how you can make the visualisation work for you e The web based visualiser is very powerful many selection and visualisation options can be accessed by right clicking in the visualiser and choosing Select Popup or by pressing the F11 key on the keyboard Control panel Select Generate Modify Lights Mode Select C Append C Exclude Molecules Builtins PDB pdb2gla__ root GrResidues G Contact Aminoacid Mainchain Calpha Solvent Ions DNA Whole residue Whole molecule 10th residue FN UPR ry ceee Lines Width j1 Cylinders Radius 0 15 Sticks Ball 030 Stick 015 Spheres Radius 1 50 S Transp 255 3 Colour Labels clear v F Solid View controls Reset Back a H l Fog D AA l Shadows e The Hermes visualiser has been tailored to work with Relibase particularly in terms of making use of the information f
8. 3 Drawing and Fusing Rings 3 1 Adding a Ring to a Blank Drawing Area e Rings may be drawn manually but the easiest way is to use the pre drawn rings to the left of the Draw window Templates Other e Ifthe desired ring is one of the four on display see above select it by clicking on its icon move the cursor into the white area then click with the left hand mouse button e Click on the Draw button to stop drawing rings e Some complex ring systems are available by clicking on Other in the Templates section to the left of the Draw window 3 2 Adding a Ring to an Atom in an Existing Substructure e Select the ring see Adding a Ring to a Blank Drawing Area Section 3 1 page 125 then click on the desired atom in the existing substructure with the left hand mouse button Relibase User Guide 125 e For example selecting a 6 membered aromatic ring and clicking on the terminal C atom in will create c y Sg C 5 C c a c 2 p ges i Ree 3 3 Fusing a New Ring to an Existing Ring e Select the new ring see Adding a Ring to a Blank Drawing Area Section 3 1 page 125 then click on the desired fusion bond in the existing ring e For example selecting a 5 membered saturated ring and clicking on one of the C C bonds in aes TA a C c will create 3 4 Creating a Spiro Fusion e Select the required ring see Adding a Ring to a Blank Drawing Area Section 3 1
9. Hydrophobic Atoms The definition of hydrophobic atoms includes almost all types of carbon atom but not cyanide or carbonyl carbon sp hybridised sulphur non ionised chlorine bromine and iodine and any 194 Relibase User Guide hydrogen atom that is covalently bonded to a hydrophobic atom effectively C H and S H Note that S H is also counted as a donatable hydrogen Number of Buried Hydrogens Acceptors Not Forming an H Bond The following descriptors e Number of buried donatable ligand hydrogen atoms not forming an H bond e Number of buried ligand acceptors not forming an H bond e Number of buried donatable protein hydrogens not forming an H bond e Number of buried protein acceptors not forming an H bond are counts of occluded hydrogen bonding atoms i e atoms that are inherently capable of participating in hydrogen bonds but are prevented from doing so because they are buried by non H bonding atoms For example a histidine ring NH would be counted as occluded if it were solvent accessible before docking but was buried by ligand carbon atoms after docking A limitation of these descriptors is that no allowance is made for intramolecular hydrogen bonding i e in the example just quoted the NH would still be counted as occluded even if it were H bonded to a neighbouring protein atom Polar non Hydrogen Bonding Atoms Polar atoms in descriptors such as Number of buried polar atoms in the ligand are atoms that have some p
10. Pseudo centres match 7 RMS match 1 98 Pseudo centres clique 8 RMS clique 1 98 Score 5 55 Protein Homology 18 60 Visualiser Automatic Visualiser Updates Load in Hermes Query cavity pdbtjus 5 Similar cavity pdb2jf0 9 Volume 2932 75 1021 12 Pseudo centres 195 30 total PDB djus 2if0 Header TRANSCRIPTION HYDROLASE CRYSTAL STRUCTURE OF THE MULTIDRUG Title BINDING TRANSCRIPTIONAL REPRESSOR QACR MUS MUSEUEUS AEE TYLEHOLNESTERASE N BOUND TO RHODAMINE 6G COMPLEX WITH TABUN AND ORTHO 7 MOL_ID 1 MOLECULE HYPOTHETICAL TRANSCRIPTIONAL REGULATOR IN QACA MOL_ID 1 MOLECULE ACETYLCHOLINESTERASE CHAIN A B FRAGMENT CATALYTIC DOMAIN Compound S REGION CHAIN B D A E SYNONYM QACR g s i REPRESSOR ORF 188 ENGINEERED YES menea Pape ENGINEERED VES oo MUTATION YES Ligands Cavity object go to cavity information page go to cavity information page e Click on the Load in Hermes link This will display both the query and the hit cavities superimposed and will also display the pseudocentres and surface patches that have been matched Another window the Cavity Controls window will also come up e You can hide the two window that appear to the left The Protein Explorer and the Contacts windows to increase the display area by clicking at the top right of each window Click on the Unassigned Surface tick boxes in the window headed Explore n
11. v Ligands y Chains _ Solvent y Packing i Schematic Show Embedded Visualiser M width 400 Height 300 Apply Hermes Controller Showin Hermes Automatic Visualiser Updates I Header SERINE PROTEASE Title X RAY STRUCTURE OF THE COMPLEX OF HUMAN ALPHA THROMBIN WITH THE INHIBITOR SDZ 229 357 Compound MOL In 1 MOLECULE Al PHA THROMRIN CHAIN AF 9491748 MOL IN 9 MOLECULE Al PHA THROMRIM CHAIN RFC 34715 27 Additionally the following buttons are present at the top of each protein entry page 4 2 Each Relibase ligand page contains 28 View PDB Header launches a new browser window with the complete header of the original PDB file Save PDB File export the protein structure in pdb file format PDB Website links to the current protein entry on the PDB homepage Attp www rcsb org pdb Bookmark add the current protein entry page to your list of favourites in your browser Relibase Ligand Pages Embedded 3D visualisation via Astex Viewer see Astex ViewerTM Section 5 1 page 42 A Hermes control panel see Hermes Section 5 2 page 47 Protein and ligand information see Protein and Ligand Information Section 4 3 page 30 Information on water structure in the entry see Water Information Section 4 4 page 31 A link to information on cavities in the entry see Cavity Information Section 4 5 page 34 Information on the secondary structure in the entry see Secondary Structure Info
12. Cavity Controls You can alsa show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1jus 5 in reli Hit CavBase Hit pdb2gyu 12 in reli Display Controls Search Setup Show Pseudocentres Query Matched PCs Hit Matched PCs Link matching PCs with lines Separate cavities Relibase User Guide 39 e Hide the surface patches for both cavities by clicking the Active Surface tick boxes in the Graphics Object Explorer e Only the matched pseudocentres will be displayed The display should look as illustrated below and shows that amongst other things Trp61 of the QacR cavity has been matched with Tyr 124 of AChE and Tyr930f QacR with Tyr341 of AChE You can get the number of a residue by clicking with the right hand mouse button on any atom of the residue and selecting Labels followed by Label by Protein Residue e If you have labelled anything remove the labels by clicking with the right mouse button anywhere in the display area background and selecting Labels followed by Do not label e Now we will examine how well the ligand from the hit cavity might fit into the query cavity You will need to activate the Protein Explorer window by selecting it from under View in the top level menu bar of Hermes Turn off the Chain tick box for the query and turn off the Ligand tick box for the Hit e The ligand from the hit cavity sho
13. Defining Cyclic or Acyclic Atoms It is possible to specify that a particular atom must be cyclic i e part of a ring or conversely that it must be acyclic i e not part of a ring In Draw or Select mode click on an atom with the right hand mouse button pick Cyclicity from the resulting pull down menu then select the required option from the second pull down menu Unspecified in this menu means the atom may be either cyclic or acyclic If the atom is already part of a ring the Acyclic option will not be active For atoms which form part of a ring i e cyclic atoms it is also possible to specify the ring size Note When a particular atom is part of more than one ring the smallest ring will be considered when testing the constraint To set a maximum limit on the ring size click on an atom with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Maximum smallest ring sizes then select the required option from the third pull down menu To set a minimum limit on the ring size click on an atom with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Minimum smallest ring sizes then Relibase User Guide select the required option from the third pull down menu To specify an exact ring size click on an atom with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Exact smallest ring sizes then select the required option
14. Microsoft Internet Explorer mE x A Protein Topology Neighbourhood Density Neighbourhood density in first coordination shell 2 98 Neighbourhood density in first two coordination shells 35 24 SAS Solvent Accessible Surface buried 78 Neighbourhood Density e This is a simple characterisation of the local degree of burial micro cavity of a water molecule based on analysing the local atom density e The descriptor reports the weighted sum of non hydrogen atoms within the first 3 7A and second 7 0A coordination shells respectively water molecules are neglected in the summation Linear cutoff functions apply between 3 3A and 3 7A and between 6 5A and 7 0A respectively SAS Solvent Accessible Surface e This descriptor represents the portion of the water sphere VdW radius 1 4A which is not covered by protein or ligand atoms It is a more refined characterisation of the degree of burial compared to the neighbourhood density For the calculation the water molecule is treated as a ligand or protein oxygen atom with all other water molecules excluded 5 6 3 Data Related Issues The following data are available for individual water molecules e Crystallographic B factor e Mean B factor of protein environment All protein atoms within a 3 3A radius are considered e Mean B factor of environment e Crystallographic occupancy e Mobility a scaled measure for the mobility of a water molecule encou
15. pdbtcsb pdbtcse pdbtezs pdbiczt pdbtczv View PDB Header Save PDB File POB Website Protein PDB Entry 1a0l astex r Ligands v Chains r Solent Packing Metals Schematic pdbidtd pdbidty pdb1dx5 Reliview Controller Show in Reliview Automatic Visualiser Updates I SERINE PROTEINASE HUMAN BETA TRYPTASE A RING LIKE TETRAMER WITH ACTIVE SITES FACING A CENTRAL PORE Compound MOL_ID 1 MOLECULE BETA TRYPTASE CHAIN A B C D EC 3 4 21 59 BIOLOGICAL_UNIT TETRAMER OTHER_DETAILS IN COMPLEX WITH AMIDINO PHENYL PYRUVIC ACID APPA A SYNTHETIC 158 hits for query bode INHIBITOR Reference NATURE 392 1998 306 Browse Hit Headers To see if any of the retrieved entries were also done by author Stubbs carry out an author search as just described on Stubbs and save these results in a hitlist as well e g Stubbs The Relibase hitlist manager can be invoked from the top level menubar and this allows easy combination of hitlists using boolean operators Thus the intersection of two hitlists can be easily generated e Click on the Hitlists button on the Relibase menubar This loads three frames into the browser below the menubar The top left frame lists the hitlists you have stored according to Set Name Type Ligand or Protein and Size number of entries in hitlist The Remove column allows hitlists to be deleted History and Last modification date and time are als
16. Cavities larger than 30004 many be slow to load in the visualiser and are unsuitable for cavity searching pdb8aat 8 pdb aat 9 Similarity Search Results 15 16 Secondary Structure Information for PDB Entry 1a4g Ligands Chains Solvent y Packing Metals Helices y Helix Vectors v Strands y Strand Vecto Ribbon y Turns reverse Va tir anti para Relibase User Guide CHAPTER 2 GENERAL FEATURES OF RELIBASE Nana kh DN 1 1 Getting Started see page 17 Starting a Search see page 19 Viewing and Navigating Search Results see page 20 Viewing Information for Individual Hit Structures see page 27 3D Visualisation of Structures see page 42 Storing Combining and Converting Search Results see page 47 Getting Started The Basics of Using Relibase e Relibase is a web based application and all its functionality is accessible via a web browser 1 2 The main Relibase page features a menubar which contains the following buttons e Home provides access to the Relibase Home Page see The Relibase Home Page Section 1 2 page 17 e Text Search Sequence Search SMILES Search Sketcher provide access to query constructor pages used to define queries and start searching the Relibase database e Hitlists used to view and combine results from previous Relibase searches see Storing Combining and Converting Search Results Section 6 page 47 e Stored Re
17. O Koch G Klebe G Schneider Proteins 74 344 352 2009 Relibase User Guide 173 174 Relibase User Guide Acknowledgements Relibase Development The following people were involved in the development of ReLiBase prior to the CCDC taking over the onward development and maintenance of the program now known as Relibase Dr Manfred Hendlich Dr Gerhard Barnickel Dr Klemens Hemm and Dr Karl Aberer Dr Ingo Dramburg e Dr Judith G nther e Dr Stefan Schmitt e Dr Andreas Bergner e Prof Gerhard Klebe e Dr Oliver Koch Third Party Software in Relibase The following third party software is used in Relibase e The FASTA package for sequence searching and alignment by Bill Pearson Attp www people Virginia EDU wrp pearson html Dr Henry Spencer s regex library Regular Expression Library e Code from the SPLASH library by Dr Jim Morris Attp www wolfman com splash html e The ligand 2D diagrams were generated using the CACTVS Toolkit by Dr Wolf D Ihlenfeldt University of Erlangen Germany http www2 ccc uni erlangen de software cactvs and ChemDraw http www camsoft com and Marvin by ChemAxon Ltd e Embedded visualisation powered by AstexViewer hitp www astex therapeutics com AstexViewer e AstexViewer also incorporates Thinlet http thinlet sourceforge net home html Other Acknowledgements e The staff of the Protein Data Bank at Brookhaven National Labora
18. O Acceptor O Donor i aO Pi You will note that the PseudoCentre points are no longer highlighted active and that the receptor surface associated with them is also no longer displayed Right click the mouse when the cursor is sitting on one of the ligand atoms Two options appear Select PseudoCentres within range of this ligand Select PseudoCentres within range of this atom With the left mouse button select PseudoCentres within range of this ligand A Select to Range window will appear Accept the default value of 4 00 by clicking on OK The Hermes visualiser will resemble the following Relibase User Guide Hermes File Edit Selection Display View Calculate Descriptors Help ng I Stereo ShowH HideH ShowUnk Hide Unk Graphics Objects Style Wireframe Picking Mode Select Atoms Gear Measurements x 90 y 90 y 90 2 90 z 90 amp gt J T zoom zoom Atom selections bdl F Highlighting M i X Xt y y Z zt X Display Movable aa Chains M All Entries A CaRace Muar W a R Ligands a Waters Metals M Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query Hit CavBase Query pdb1bxo 1 in reli None Display Controls Search Setup Use any of the methods below to select pseudocentres PCs in the Query cavity to be searched for fattolicking an PC
19. Once all ligands in the protein structure have templates the data processing continues calculating additional information such as crystal packing around the binding sites and cavities in the protein Finally the data are added as a new entry to the Relibase database Relibase User Guide 157 158 Several features have been implemented to minimise the amount of manual intervention required The strictness of data processing in terms of which fields are required and the syntax allowed in the PDB file can be customised in the relibase_processing conf file So for example if none of your in house structures contain an AUTHOR record you can set the author_required flag to false Alternatively if you insist on the AUTHOR record being present in your in house structures you can set the author_required flag to true If you do want to avoid having to validate ligand templates mid processing there is an option to supply ligand templates with the PDB file at the beginning of the process This option is particularly powerful if you want to process a backlog of thousands of in house structures Another powerful feature when dealing with legacy structures is the possibility to set up a synonyms file So for example if your in house structures use multiple naming conventions for water HOH H20 TIP these can all be converted to the standard format expected by Relibase HOR using the synonyms file Similar synonyms can be useful for
20. The secondary structure module allows for searching for residues that are in kinks or adjacent to kinks To define kink properties deactivate the Ignore strand kinks tick box and select from the resultant options e Use the Reset button to return the settings back to their original display 13 5Defining Turn Properties e The secondary structure module also provides the ability to search for specific turns in PDB 152 entries For turn searches automatic assignments from the following publication are always used Turns revisited A uniform and comprehensive classification of normal open and reverse turn families minimizing unassigned random chain portions O Koch G Klebe Proteins Structure Function and Bioinformatics 74 353 367 2008 DOI 10 1002 prot 22185 All types of turn described in the above publication can be searched Relibase User Guide Secondary Structure Constraints xi Helices Sheets amp Strands Turns 2 Residues 3 Residues 4 Residues 5 Residues 6 Residues normal open reverse Turn Type v Ignore Turn Type g r Ou Oma Omb Ow OW Ov CI No Typ All _ Must not be one of selected turn typ normal a turn Position v Ignore Position First cond _ Third _ Fourth _ Last Reset Reset Constraints Helices Sheets amp Strands Turns Ok Cancel Turns are irregular
21. User Guide 191 192 Relibase User Guide APPENDIX F Calculating Descriptors Computational Details Acceptor Atoms see page 193 Atom Types see page 193 Buried Atoms see page 194 Donatable or Polar Hydrogens see page 194 Hydrogen Bonds see page 194 Hydrophobic Atoms see page 194 Number of Buried Hydrogens Acceptors Not Forming an H Bond see page 195 Polar non Hydrogen Bonding Atoms see page 195 Rotatable Bonds see page 195 Solvent Accessibility see page 195 Solvent Inaccessible Ligand Surface Area see page 195 Sphere Accessible Volume see page 196 Acceptor Atoms An acceptor atom is any atom capable of accepting a hydrogen bond This includes almost all oxygen and nitrogen atoms but with the exceptions of trigonal planar R3N and quaternary R4N nitrogens since these have no free lone pairs Oxygen atoms in conjugated environments e g furan oxygen and the central nitrogen of an azide group are counted as acceptors though this is questionable Atom Types Some options in the Relibase visualiser allow specification of Sybyl atom types devised by Tripos Inc http www tripos com Amongst the most common types are H hydrogen Cu sp carbon C 2 sp carbon C3 tetrahedral carbon C ar aromatic carbon N 1 sp nitrogen N 2 two coordinate non linear nitrogen N 3 pyramidal trigonal nitrogen N p13 planar trigonal nitrogen N am amide nitrogen
22. Wed May 10 17 10 33 2006 This page will be updated in 15 seconds Current position in the search Number of cavities compared Py Number of cavities skipped due to filters 2 Number of proteins processed 0 far Search settings used maximum permitted homology between proteins 20 100 minimum score to save number of solutions to save search all entries in the entire database Click here to view all search results Click here to view current results for this query e The display will be updated every 15 seconds so that you can monitor the progress of the search Note that the number of cavities to be searched will invariably exceed the number of proteins since many proteins contain more than one cavity To view the results so far click on the hyperlink Click here to view current results for this query This will take you to a page listing the hits in descending order of similarity see Browsing the Results of a Search Section 4 6 page 113 Clicking on any hit will load it and the query into Hermes e Cavity similarity searches can take many hours to run If you want to stop a search before it has finished click on Finish this query now Any hits already found will be kept 4 6 Browsing the Results of a Search e Ifyou start a search and then keep your Relibase session open at the search progress page see Monitoring Search Progress Aborting Searches Section 4 5 page 112 you will on completion automatical
23. a E Relibase User Guide 109 e Because pseudocentres can also be selected and deselected in other ways e g by clicking on them it is possible to generate a state in which some pseudocentres of a given type are selected and some are deselected This is indicated by a tick box on a grey background e g Display Controls Search Setup Use any of the methods below to select pseudacentres PCs in the Query cavity to be searched for Jettolicking on PCs nghtclick on objects to pick nearby PCs clicking on the tick boxes below Use the pulldown options below to sort the pseudacentres by different criteria 1st Sort Backbone or Sidechain 2nd Sort Pseudocentre Type v 3rd Sort Residue Type v Column 1 PseudoCentres Backbone E Acceptor a O Donor Si Pi aM ALA O ASP e O CYS dechain Metal Acceptor ASP O GLN a M Aliphatic a O Aromatic a O Donor m K Spam i e Once you have selected the pseudocentres you want to search for hit the Search button at the bottom right of Hermes This will open a browser page where you can set some other search options and then start the search 4 4 Setting Search Options and Starting the Search e Once you have specified the search query i e hit the Search button in the Search Setup pane of the Cavity Controls pane see Selecting the Pseudocentres to be Searched For Section 4 3 page 108 you will be taken to
24. and Bioinformatics 74 353 367 2008 DOI 10 1002 prot 22185 Relibase User Guide 35 A full description of all turn types is available as supplementary material for the above reference e Attp archiv ub uni marburg de diss z2008 0730 This reference also includes the amino acid propensities for all turn types e Publications on SHAFT and the secondary structure module in Relibase are currently in preparation e Secondary structure assignments are classified into the following e Turn Assignment see page 36 e Turn Types see page 36 e SHAFT Assignment of Helices see page 37 4 6 3 Turn Assignment e A diverse subset of 1903 chains was used as a training set for assigning of turn types Initially turns were identified in the training set e Each sequence of up to 6 residues in each peptide chain was extracted and analysed to identify close contacts between the terminal residues Several types of contact were identified Hydrogen bonds were deemed to be present if the DSSP function Kabsch and Sander in house implementation indicated their presence Hydrogen bound turns were then classified as either normal or reverse dependent on the direction of the hydrogen bond with respect to the direction of the peptidic sequence Additionally open turns were identified where the Ca Ca distance between the terminal residues in the sequence was less than 10A Sequences that fell into one of these classifications was deemed
25. and rescale the complete query such that it will fit into the sketcher window select View from the top level menu followed by AutoFit from the resulting pull down menu Scaling Queries It is only possible to scale complete fragments not a collection of atoms which form part of a fragment Select the fragment s to be scaled and then clicking on one of the corners drag the mouse till the fragment is the appropriate size In any mode use the mouse scroll wheel to adjust the scale of the contents of the drawing area Alternatively use the zoom buttons in the View Controls section to the left of the Draw window 135 Relibase User Guide 8 3 136 View Controls To automatically resize the complete query to the maximum minimum or default zoom level select View from the top level menu Zoom from the resulting pull down menu then select the required option from the subsequent menu To automatically rescale and move the complete query such that it will fit into the sketcher window select View from the top level menu followed by AutoFit from the resulting pull down menu Rotating Queries It is only possible to rotate complete fragments not a collection of atoms which form part of a fragment Select see Selecting Atoms Section 2 7 page 123 the fragment s to be moved While keeping the Control button depressed use the left hand mouse button to rotate the fragment s Release the left hand mouse button at the desired po
26. page 45 e Secondary structure display see Secondary Structure Display Section 5 1 4 page 46 5 1 1 Appearance of AstexViewer on a Protein Information Page Protein Entry 1a1m 7 z Chains _ Solvent y Packing v Schematic Show Embedded Visualiser M Width 600 Height 450 Apply The display can be rotated translated and scaled as previously described see Astex ViewerTM Section 5 1 page 42 e A number of check boxes beneath the display can be used to control the view e Ligands displays or hides ligands e Packing displays or hides any crystal packing present in the protein e Chains displays or hides protein chains e Metals displays or hides metal atoms e Solvent displays or hides solvent molecules e Schematic displays or hides the protein cartoon display Relibase User Guide 43 Note the schematic is assigned by Astex Viewer and not Relibase nor the PDB e Whether or not Astex Viewer is displayed is controlled via the Show Embedded Visualiser tickbox The dimensions of the viewer size default is 800 by 600 may also be modified to fit user requirements by typing new dimensions into the Width and Height windows then hitting Apply 5 1 2 Appearance of AstexViewer on a Ligand Information Page Ligand DIU_2001 A PDB entry 2bxl Chemical name 2 HYDROXY 3 5 DIIODO BENZOIC ACID v Ligands v Chains Solvent l Schematic Show Embedded Visualiser M Width 600 H
27. see Performing a Keyword Search Section 3 1 page 58 3 Keyword Searches 3 1 Performing a Keyword Search e Click on the Text Search button in the Relibase menubar e Select the Keyword option in the Search Type box By default the Search Field will be HEADER TITLE COMPND and SOURCE Records A number of options will become available e Type the required text string into the Search String box Relibase PDB Entry Code SI Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Text Search relibase 3 0 0 rcs Search Type Keyword z Search String bovine trypsin Match whole words only I Use regular expressions Keyword Search Options Search Field HEADER TITLE COMPND and SOURCE Records x Filter Search Options Minimum Year Maximum Year Minimum MolVVeight Maximum MolWeight Highest Resolution A 1 0 Lowest Resolution A Structure Method Filters X ray M NMR M Use Hitlist Select existing hitlist z Save in Hitlist Overwrite Existing Hitlist I Submit 58 Relibase User Guide e Use the Match whole words only tick box to gain further control over the results obtained e Regular expressions may be used for example e trna guanine transglycosylase the use of quotes means that a match will be found for the entire string e trna the use of matches the start of the string In this case the query would match trna synthetase but not
28. see Similar Protein Chain Searches Section 8 page 81 can also be initiated from the resulting page Information on water structure in the entry can be accessed by clicking on the Water Information button see Water Information Section 4 4 page 31 Information on cavities in the entry can be accessed by clicking on the Cavity Information button see Cavity Information Section 4 5 page 34 Relibase User Guide e Information on the secondary structure in the protein can be accessed by clicking on the Secondary Structure Information button see Secondary Structure Information Section 4 6 page 35 e Customisable content and hyperlinks to external resources can also be added see the Relibase Installation Notes Appendix B http www ccdc cam ac uk support documentation relibase 4 4 Water Information 4 4 1 Information on Water Structure Information about the water structure for each database entry is accessed by clicking on the Water Information button at the bottom of either the Relibase protein entry page see Relibase Protein Entry Pages Section 4 1 page 27 or the Relibase ligand page see Relibase Ligand Pages Section 4 2 page 28 General Information on Water Structure in Entry pdb1a3e Water models 1 Water molecules 206 Water molecules per residue 0 741 Average B factor 34 921 A Standard deviation of B factors 10 231 Dubious Water Molecule Criteria for Notific
29. 113 700 c 138 100 alpha 90 000 beta 90 000 gamma 120 000 Spacegroup P3121 Resolution RFactor Deposition Date Ligands THA in pdb acj Water mediated protein ligand contact paths 2 Protein Chains pdbtacj A Nucleic Acids No nucleic acids for this entry Secondary Structure Secondary Structure Information Waters Water Information Cavities Cavity Information For a typical entry the following information is given 30 A summary of the textual bibliographic and crystallographic information for this protein entry including Header Title Compound Reference Author s Source Method Crystal Resolution RFactor and Deposition Date Click on any author s name to run an author search see Author Name Searches Section 3 2 page 59 on that name A Caveat record is present for PDB entries that contain information under the CAVEAT section i e are considered to be in error by the PDB in their PDB file In the example above the structure contains only one ligand and binding site a tacrine molecule Clicking on the 2D chemical diagram of the tacrine molecule will link to the ligand page see Relibase Ligand Pages Section 4 2 page 28 for this ligand binding site The amino acid chains in the structure are listed in the example above there is only one chain The protein chain sequence can be viewed by clicking on the Chain Identifier hyperlink Searches for similar chains
30. 3 and pdb148I 1 Pseudo centres match Pseudo centres clique 0 79 Protein Homology 12 10 To reload comparison Query cavity pdb1xie 3 Similar cavity pdb1481 1 Volume A 2701 50 Pseudo centres 190 total ISOMERASE INTRAMOLECULAR OxIDOREDUCTSE Header HYDROLASE O GLYCOSYL LYSOZYME E C 3 2 1 17 MUTANT WITH THR 26 REPLACED BY GLU CYS 54 REPLACED BY THR D XYLOSE ISOMERASE E C 5 3 1 5 COMPLEXED AND CYS 97 REPLACED BY ALA WITH 1 5 DIANHYDROSORBITOL PH 7 4 T26E C54T C97A COMPLEXED WITH SUBSTRATE CLEAVED FROM CELL WALL OF ESCHERICHIA COLI Compound Ligands Cavity object go to cavity information page Relibase User Guide 115 e The search program uses a clique detection algorithm to produce a preliminary mapping of query and hit cavity pseudocentres the entries Pseudo centres clique and RMS clique refer to the number of pseudocentre pairs in this match and their RMS deviation respectively Additional pseudocentres may be added to this preliminary mapping or pseudocentres may be dropped from it and Pseudo centres match and RMS match give the number of pairs and RMS deviation for this final mapping All the other information in the tables should be self evident e Clicking on a protein identifier or a ligand diagram will take you to the relevant Protein or Ligand Information page 4 7 Managing Search Results e To see a list of all the cavity similarity searches you have run click on the
31. AstexViewer Manfred Henalich 1994 7999 and Cambridge Crystallographic Data Centre 7999 2008 WSC WSC The Home Page provides the following options e Click on the CCDC logo to go to the CCDC web site http www ccdc cam ac uk e Enter a PDB code into the PDB Entry Code box and click View for quick access to a protein of interest e Click on the link Install 3D Visualization Software to download Hermes the software required to visualise Relibase entries in 3D e Click on the Client Workspace Administration to access other client workspaces available for unlimited licenses only The workspace username and the databases that are currently loaded are displayed at the bottom of the page e Click on the link In house Database Building Tool to build proprietary database s e Use the Cavity Similarity Results link to hyperlink to previously saved cavity hitlists e Click on support ccdc cam ac uk to email us with any problems enhancement requests etc Relibase User Guide 2 Starting a Search The buttons on the Relibase menubar provide access to query constructor pages In these pages you can define queries and start searches Note that the PDB Entry Code box can be used for quick access to a specific PDB code of interest Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Stored Results e Text Search provides access to e Searches on protein entry code see PDB Entry Code Searches Sec
32. Cavity Similarity Results hyperlink on the Relibase home page Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Relibase Homepage Relibase 3 0 0 server rc2 Relibase A program for searching protein ligand databases Install 3D Visualization Software Client Workspace Administration In house Database Building Tool Cavity Similarity Results Comments bugs queries to Cambridge Crystaliographie Data Centre 72 Union Road Cambridge CB2 7EZ a United Kingdom tel 44 1223 336022 www http www cede cam ac uk S e mail support cede cam ac uk Current workspace henderson Databases currently loaded gregtest reli ssetest Pef EL KA f ry Diagram generation in Relibase is powered by ChemAxon s Marvin Software E chemAxon m astex Embedded visualisation in Relibase is powered by AstexViewer e A link to cavity similarity search results is also available via the Stored Results tab 116 Relibase User Guide e Hyperlinks to the cavity similarity search results list can be found on any cavity information page see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 e g Cavities in PDB Entry 1xie CAM pdb 1xie 1 308 500 CAV pdb 1xie 2 1599 000 CAV pdb 1xie 3 2701 500 Similarity Search Results e An example list of cavity similarity searches is Comparisons
33. HAREL LSILMAN Source TORPEDO CALIFORNICA X RAY DIFFRACTION Cell a 113 700 b 113 700 c 138 100 alpha 90 000 beta 90 000 gamma 120 000 Spacegroup P3121 Resolution Resolution 2 800 RFactor 19 5 Deposition Date 18 8 1993 Ligands THA in PDB Entry 1acj Water Mediated Protein Ligand Contact Paths 2 Protein Chains pdblacj Show sequence Submit Search for Similar Chains foo 8 Minimum Sequence Identity foo 8 Maximum Sequence Identity D Save in Hitlist I Overwrite Existing Hitlist M Show Ligands WaterBase Water Information CavBase Cavity Information Detailed information on the water structure in the entry can be accessed by clicking on the Water Information button Detailed information about cavities present in the protein are found by selecting the Cavity Information button It is also possible to perform cavity similarity searches by clicking on this button Additionally at the top of every protein entry page there are several buttons e View PDB Header launches a browser window with the complete header of the original PDB file e Save PDB File allows the entire PDB file to be downloaded and saved PDB Website links to the current entry on the PDB website http www rcsb org pdb Bookmark allows you to save the page so you can return to it later Open Hermes by selecting the Visualise button at the top of the page and inspect the structure found Familiarise yourself wit
34. Section 1 2 page 120 Ensure the molecule type is set appropriately i e protein ligand or water see Setting Molecule Types Section 1 3 page 120 Move the cursor into the white area of the drawing window Press down the left hand mouse button move the cursor while keeping the mouse button depressed and then release the button This draws a bond using the appropriate element and bond types see Changing the Current Element Type Section 4 1 page 121 or Changing the Current Bond Type Section 5 1 page 125 To draw bonds of fixed length select bond length from the Options menu and tick the Fixed box The length Standard Half Double can be selected using the radio buttons To draw bonds which are fitted to a grid select Grid in the top level View option and click on the specific grid required Horizontal triangles Vertical triangles or Square Select No Grid to remove The GridSize slider alters the Grid Size To change its orientation type the rotation angle into the box Drawing an Isolated Atom Ensure you are in Draw mode see Modes in the Drawing Window Section 1 2 page 120 Ensure the molecule type is set appropriately i e protein ligand or water see Setting Molecule Types Section 1 3 page 120 Move the cursor into the white area of the drawing window Click the left hand mouse button and release it again without moving the mouse Drawing a Bond from an Existing Atom Ensure you are in Draw mode see Modes in
35. Steps Required Set up a cavity hitlist so that we only search a subset of the database e Set up the search query specifying which parts of the query cavity we want to find matches for e Specify search options and run the search e View a hit cavity together with the query cavity in the 3D viewer using various viewer options to assess how well the two match e Experiment with some other viewer settings 3 1 3 The Example The S aureus multi drug binding repressor protein QacR is known to bind several drugs and is relevant to the mechanism of multi drug resistance in this organism D S Murray M A Schumacher and R G Brennan Crystal Structures of QacR Diamidine Complexes Reveal Additional Multidrug binding Modes and a Novel Mechanism of Drug Charge Neutralization J Biol Chem 279 14365 14371 2004 e In this tutorial we will use CavBase to search for other ligands that might bind to this protein 3 2 Setting Up a Hitlist e Open Relibase e When we do the cavity similarity search we could if we wished search the whole database but this would take a long time To speed up the tutorial we will therefore search on a subset that just contains the esterases There is no good reason for this except that as we will see there 1s at least one interesting hit in this subset e To do this we must first do a text search for esterase and save the hits in a protein hitlist e So hit the Text button select Keyword Search fro
36. This page will be updated every 15 seconds The search will take a few minutes to run e Once the search has run you will be presented with a list of the hits that have been found By default they are ranked by similarity score so the cavities that match the query best will come first Many of the hits are Acetylcholinesterase In the second page of results should be the hit pdb2jf0 9 This has very low sequence homology with the query 18 6 Seven of the pseudocentres that you included in the search query were matched to pseudocentres in the pdb2jf0 9 cavity Note This cavity search is for illustrative purposes only The scores and number of pseudocentres for the best hits matched in this search are quite low and so cavities that are very similar to the cavity in QaCR have not been found 36 Relibase User Guide Search Results 1 15 of 100 Normalised Matched Protein Cavity Score RMS Header itle Score Centres Homology pdb2vi0 12 0 443 174 HYDROLA CRYSTAL STRUCTURE OF N TERMINAL DOMAIN OF ACYL COA THIOESTERASE 7 0 772 HYDROLA CRYSTAL STRUCTURE OF MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX WITH HI 6 MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX 1 055 HYDROLA CRYSTAL STRUCTURE OF MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX WITH OBIDOXIME pdb2jey 12 CRYSTAL STRUCTURE OF MOUSE dchas ii JEA 0 92 EJ HYDRO ACETYLCHOLINESTERASE COMPLEXED WITH CHOLINE NON AGED FORM OF MOUSE ACETYLCHOLINESTERASE pdb2c0g 1
37. a browser page looking something like this 110 Relibase User Guide Similarity Search for Cavity pdb1a8t 1 using 39 Pseudocentres Parameter Setting Select an existing hitlist The entire database x Search name fearch_pab1 adt 1_39 Maximum permitted homology between proteins 00 0 Minimum ligand size N atoms B _ Minimum score to save T Number of solutions to save top N ho FCti lt i SS S Maximum resolution permitted Search priority nice value 10 highest 19 lowest priority ce scoring function 1 Select a Scoring Function scoring function 2 C scoring function 3 Start Search e This page can be used to set a variety of search options as follows Use the Select a cavity hitlist pull down menu to specify whether you want to search the whole database pick The entire database from the pull down menu or a subset In order to search a subset you must have previously created a hitlist defining that subset see Searching a Subset of the Database Section 4 4 1 page 112 if you have done this simply pick the relevant hitlist from the pull down menu Type in a name for the search Specify the maximum permitted homology as a percentage This can be used to increase the novelty of the search results by rejecting hit cavities from proteins that are similar in sequence to the query cavity protein Such hits are often trivial and can be found more quickly by sequence based similarity search me
38. a pull down menu from which you can choose to Display matching atoms only Display matching chains or Display entire binding site Keep the default of Display matching atoms only for this example Hit Start to run the search When the search is complete the results are displayed in the main Relibase window Click on the View in Hermes button to view the results in Hermes Relibase User Guide e Select the Histogram s link to find which contact distance is the most common When you have defined any geometrical parameters in your query histograms for these parameters are generated automatically and can be viewed by clicking on the Histogram s link in the bottom left frame of the Relibase ligand browser Histogram Label D1 Distance Distribution Slice Size 0 2 A Update Distance Distribution 425 170 145 ie l 3 Lo a 3 32 34 24 26 28 Total Distribution Size 814 e We can also inspect the preferred angle between the planes of the two moieties Relibase User Guide 27 The histogram s can be exported in a comma separated value csv file for analysis in third party software and hyperlinks to structures from specific histogram bins are available 2 3 2 Protein Protein Interaction Searching The Relibase sketcher also allows you to set up protein protein interaction searches without specifying any ligand substructure Centroids of selected atoms can be defined as a means to specify more complex geometrical s
39. and bond typing assigned by Relibase match_against_pdb_templates true false If this flag is set to true Relibase will attempt to use ligand templates from the main Relibase database as input templates based upon matching the ligand three letter code and substructure matching repair_incomplete_conect_records true false The default option for this flag is set to false In this case Relibase will only try to guess which atoms are connected to each other a separate concept from determining atom and bond types if there are no CONECT records present If there are CONECT records present these will be used to determine which atoms are connected to each other However if a PDB file for some reason or other contain ligands that only have part of their connectivity explicitly defined by CONECT records Relibase will not identify these bonds This can be overcome by setting the repair_incomplete_conect_records flag to true However bear in mind that this can lead to non bonded atoms becoming incorrectly connected to each other if they have coordinates that are anomalously close to each other pdb_templates_exceptions_file filename This option can be used to specify ligand three letter codes that should always be matched against templates in the main Relibase database even if the match_against_templates option is set to false Simply list the three letter codes of interest on new lines in the file For example GOL Relibase User Guide ICA
40. and one Ca atoms that is involved in two adjacent kinks in the other case 13 4Defining Sheet and Strand Properties e Within the Sheet amp Strand Properties pane it is possible to constrain residues to lie in sheets strands or kinks with given properties The Sheets amp Strands tab is subdivided into three panes e Sheet Properties e Strand Properties e Kink Properties Sheet Properties 150 Relibase User Guide Helices Sheets amp Strands Turns Sheet Properties StrandProperties Kink Properties Type v Ignore Sheet Type E A t O Allow Parallel Sheet Allow Mixed Position v Ignore sheet position a id O v Middle Strand _ All E Other id Sheet dimensions Minimum Size O Maximum Size 1 ooo Reset e Sheets can be constrained so that one searches for types of sheet for specific strands in a given sheet The first strand in a sheet is defined as the first strand that one comes to along the sequence as one traverses from the N terminus residue of the sequence to the C terminus e Specific sheet Types and Positions can be specified by deactivating the Ignore Sheet Type and Ignore sheet position check boxes Sheet types can be constrained so that a search only returns sheets that are parallel anti parallel or mixed e Sheet sizes can be constrained so that sheets containing a defined number of residues are searched for Strand Properties
41. aspartyl trna synthetase e matches any character e causes the resulting expression to match 1 or more repetitions of the preceding expression e g ab will match a followed by any non zero number of bs it will not match just a e square brackets are used to indicate a set of characters Characters can be listed individually or a range of characters can be indicated by giving two characters and separating them by a hyphen For example akm will match any of the characters a k m or a z will match any letter Note regular expression searching is not supported for ligand entry code searches see Ligand Code Searches Section 3 4 page 62 e Various additional Options are available for all text based searches However not all options are available for all searches see Options for Text Based Searches Section 3 5 page 64 e Hit the Submit button to start the search e The results are presented as a browsable list of Relibase entries The text that makes up the query will be highlighted in red in the Header Title Compound and Source fields e The search results can be saved to a hitlist after the search has been run using the Save in Hitlist button in the bottom left hand frame of the results window 3 1 1 Hints for Keyword Searching in the Header Title Compound and Source fields e The searches are not case sensitive e The searches are based entirely on the HEADER TITLE COMPND and SOURCE records available i
42. assignment by unchecking the Use original PDB helix assignments check box Helix Properties Helices Sheets amp Strands Turns Helix Properties Terminus Properties I Kink Properties Helix Type v Ignore Helix Type Right Handed Helices E O O Left Handed Helices q Other C O Helix Length Minimum Length oH Maximum Length 1 000 Reset k e To define helix properties deactivate the Ignore Helix Type check box It will then become possible to select Right Handed Helices Left Handed Helices and Other properties via their individual check boxes 148 Relibase User Guide e The minimum and maximum helix length can be defined in the Helix Length section of the panel Use the Reset button to return the Helix Properties settings to their original display e Note the SHAFT assignment only contains right handed helices 3 9 a and p helices Terminus Properties Helix Properties Terminus Properties Kink Properties N Terminus i Ignore N Terminus Properties m m n A 0 C erminus Ignore C Terminus Properties ON El O Ej Reset e Use this tab to specify the properties of a residue with respect to the N or C terminus e The N cap and C cap residues are the residues at either end of a helix with the N one N two etc being steps along from the N cap residue so the N one would be the residue in the helix adjacen
43. button You will be given a choice of three options in the pull down menu to the right These are respectively Display matching atoms only Use this option to superimpose only those substructure atoms drawn in the Sketcher query page Display matching chains Use this option to display the complete ligand structures after superimposition Note that complete residues for any protein atoms in the query are shown as well as complete ligands Display entire binding site This option clearly displays all the atoms ligand and others within 6A of each superimposed ligand After choosing the appropriate option above the search is run by hitting Start Once the search completes the superposition is loaded into the embedded visualiser Astex Viewer or can be loaded into Hermes via the Show in Hermes button The superposition can be re loaded using the Hit Superposition hyperlink in the bottom left frame Note using the Display entire binding site option can very rapidly lead to an unintelligible display as the number of superimposed ligands increases The complexity of the display is also affected by the degree of symmetry of the query Note also that this option is only available for queries containing ligands If you would like to run a search in the background without the sketcher Results tab being updated activate the Run batch search with name tickbox enter a search name then start the search Batch search results can be viewed as the search is
44. can be hidden from view in subsequent recalculations by clicking off the relevant checkbox in the Protein flexibility area at the base of the table e Each numeric entry in the Mutations and Insertions column links to an expanded list that provides further information Relibase User Guide 93 Mutations and Insertions Reference chain PDB 1fax A Reference ligand DX9_1_pdb1fax_1 Protein chain PDB 1bbr E Mutations and Insertions Reference Residue Mutated to Superimposed Residue PHE 174 ILE 174 GLN 192 GLU 192 ALA 221 ASP 221 ILE 227 PHE 227 Insertion in reference GLU 97 Insertion in reference THR 98 e Clicking on the header to column five links to a concatenation of the mutation insertion data for each relevant chain All chains are represented 9 4 6 The Analysis Table Ligand Overlap e The sixth column in the table gives the percent of ligand overlap between the ligands in the reference and superimposed chains The first figure is the percent overlap in terms of reference ligand volume the second figure is the percent overlap in terms of superimposed ligand volume The reference ligand is always the ligand that was originally used to set up the superposition analysis To change the reference ligand it is necessary to start from a different ligand page The column can be hidden from view in subsequent recalculations by clicking off the relevant checkbox in the Ligand binding site area at the base of the table e Ea
45. conformation with a hydrogen bond between CO and NH n c A distorted or open turn conformation lacking a hydrogen bond with a Ca Ca n distance lt 10A j f jj jj Different turn conformation a reverse turn with 3 residues b normal 8 turn 4 residues c open B turn 4 residues magenta hydrogen bonding yellow Ca Ca distance e The inner residues of a normal or open turn are those turns that lie within the described hydrogen 60 bonded ring Thej andy angles of these residues and the additional L angles are used for clustering Relibase User Guide 5 4 The Example e In 2000 the structure of a new kinase inhibitor was published which when crystallised in its receptor complex 1fpu parent enzyme ABL Kinase was shown to have a distinctly different binding mode from those kinase inhibitors known previously Hitherto inhibitors were found to act by direct competitive replacement of a unique essential substrate ATP at a very highly conserved site and with an equally conserved set of interactions This mimicry of highly polar ATP would clearly lead to problems of inadequate specificity and often a difficulty in being able to make new inhibitors as lipophilic as other pharmaceutical considerations might prefer e The lfpu X ray showed that this inhibitor occupies a new allosteric binding pocket spatially distinct from the ATP hinge region The image below shows a Relibase superposition of the lfpu structure with
46. csv comma separated value file Information content of this file includes PDB code ligand compound name number of heavy atoms empirical formula and the ligand SMILES code If you have more than one hitlist of a given type e g Protein Ligand or Cavity these can be added or removed from other sets of the same type and saved see Viewing and Editing Hitlists Section 6 4 page 51 Deleting Hitlists Click on the Hitlists button on the Relibase menubar This loads three frames into the browser below the menubar The top left frame lists the hitlists you have stored see Viewing and Editing Hitlists Section 6 4 page 51 Click on Delete in the column labelled Remove to remove a hitlist Loading XML Format Hitlists It is possible to read in any XML format hitlist saved out from a previous search Click on the Hitlists button on the Relibase menubar This loads three frames into the browser below the menubar In the bottom left frame there is also a Read Hitlist option see Combining and Translating Hitlists Section 6 5 page 53 To read in an XML list select XML from the Format pulldown click on the Browse button next to this select the appropriate file using the file browser and click Submit to load the hitlist Loading PDB Format Hitlists It is possible to read and save a PDB plain text listfile as a new hitlist Click on the Hitlists button on the Relibase menubar This loads three frames into the browser below the me
47. defined when drawing a substructure in the Draw window These objects can then be used for computing geometric parameters e g the distance between two centroids 10 1 Valid Geometric Objects Valid objects are e Centroids e Vectors e Planes 10 2 Defining Geometric Objects e Open up the Geometric Parameters dialogue box by clicking on the Add 3d button in the Draw window This button will only be active when there is a substructure in the drawing area e Select the atoms or existing objects in the Valid objects list that are needed to calculate the new object by clicking on them with the left hand mouse button click again on an atom to deselect e As the number of selected atoms varies the dialogue box will list the Valid objects that can meaningfully be defined e Hit the appropriate Define button in the dialogue box e g next to the word Centroid to define a centroid e The defined object is listed in the box labelled Defined Objects e In the example below the centroid of an indole substructure has been defined 138 Relibase User Guide File kal Options Help Modes Query Sketcher 3d Parameters Click on atom to add to selection F Edit 3D Parameters o Bd To construct a parameter object Select atoms in main window Templates Current selection H CENTI Valid parameters Defined paramete View Controls Valid objects Defined objects ENT1 Delete Done
48. house PDB files do not strictly adhere to the PDB file format in particular they may not contain all the PDB records Relibase expects them to have In these cases the user will be prompted to add the required field to the PBD file However this can become cumbersome if all of the in house structures are lacking that particular record Relibase therefore allows the user to define the strictness of the data processing in terms of which fields are required and the syntax allowed in the PDB file This customisation is defined in the relibase_processing conf file located in the SRELIBASE_ROOT processing directory e header_required true false This flag determines whether the HEADER record is required or not e title_required true false This flag determines whether the TITLE record is required or not J e compound_required true false This flag determines whether the COMPND record is required or not ii e source_required true false This flag determines whether the SOURCE record is required or not e method_required true false This flag determines whether the EXPDTA record is required or not e author_required true false This flag determines whether the AUTHOR record is required or not e reference_required true false This flag determines whether the JRNL record is required or not Relibase only parses the TITL and REF sub records of the JRNL records
49. in the pull down menu e Use the tick boxes in the resulting dialogue to specify which components you want to write out specify an output file name then hit Save e g Save Cavity Query Objects to Save JV Ligand s IV Chain s J Solvent P Metal lons Hit Objects to Save JV Ligand s V Chain s J Solvent J Metal lons Look in Sg C z G ee EEE Documents and Settings C3 flexim NVIDIA Program Files quarantine Oo temp unzipped Windows Update Setup Files WINNT XWIN32 File name i xie_1epz mol2 File type All Mol2 files mol2 x Cancel 6 Building In House Cavity Databases Cavity information is generated for proprietary structures by default when the structures are processed to an in house database These data are then searchable when using the Cavity Information Module Further information on producing proprietary databases is provided elsewhere see CHAPTER 6 CREATING IN HOUSE DATABASES Section page 157 118 Relibase User Guide CHAPTER 5 USING THE RELIBASE SKETCHER Sketcher Basics see page 119 Fundamentals of Drawing see page 122 Drawing and Fusing Rings see page 125 Atom Properties see page 127 Bond Properties see page 131 Using Substructure Templates see page 133 Substructure Display Conventions see page 134 Moving Scaling Rotating and Duplicating Substructures see page 135 Reading Saving and Deleting Q
50. in the top level menu then Save Query in the resulting pull down menu This will open the Save Relibase query pop up window Save Query x Queryname Owner Filename OE Access _Last Modified _ boron xml henderson Writable Readable by all 05 20 56 Wed 27 Aug 08 indole xml henderson Writable Readable by all 05 19 52 Wed 27 Aug 08 naphth xml henderson Writable Readable by all 05 21 36 Wed 27 Aug 08 jtryp_typxml henderson Writable Readable by all 05 20 24 Wed 27 Aug 08 e Enter a query name and hit the Save button To make the query readable by all users select Yes in the subsequent window e Saved queries can be read back into the drawing area by selecting File in the top level menu then Read Query in the resulting pull down menu and selecting the saved query from those appearing in the resultant dialog box e To delete a query select File in the top level menu then Load Delete Query in the resulting pull down menu select the query from those appearing in the resultant dialog box and hit Delete It is not possible to delete queries that are owned by other users Queries may be pasted from third party sketchers in MOL SDFile format via the Paste Query from System Clipboard in the Edit menu Relibase User Guide 137 Note for ISIS Draw it is necessary to first enable the Copy Mol Rxnfile to the Clipboard option under ISIS Draw Settings General 10 Geometric Objects Geometric objects may be
51. is Select existing hitlist until a hitlist is selected the entire set of databases will be searched Use the Save in Hitlist option to save the similar binding site search activate the Overwrite Existing Hitlist tick box to overwrite an existing hitlist e Similarly Use Databases allows you to select which database or combination of databases is searched The default setting is to search All databases Relibase User Guide 85 e Start the search for similar chains by clicking on the Submit button e The results are displayed as a table of chains ranked according to their sequence identity relative to the reference chain and their Smith Waterman score this is a combined measure taking into account the alignment identity and the longest sequence of matched amino acids e g in the screenshot below the alignment identity of the top entry is 100 the sequence length is 537 amino acids and the Smith Waterman score is 4407 7 e Note the Fasta sequence search finds the 1000 best sequence matches in the sequence files then filters the resulting chains on homology When searching for low homologies the required chains may not be within the 1000 best sequence matches found by Fasta Relibase PDB Entry Code Sequences Similar to PDB Lacj A retibase 3 1 0 93 Sequence s found with 99 0 100 0 Sequence Identity to Protein Chain PDB 1acj A Radius of Sphere Around Ligand A 6 0 Use entire protein For Superposition Use Binding Sit
52. of column seven links to a table which tabulates under each water in the reference structure that is relevant the details of the corresponding conserved waters in all the superimposed chains If a water is displaced by a ligand in one or other of the superimposed chains then this information is also tabulated A link is available for the appropriate ligand page ConservediDisplaced Waters Reference chain PDB 1acj Reference ligand THA_999_pdb1acj_1 Key Grey conserved Blue displaced Reference Water 634 Temperature Factor 24 0604 Conserved 10 Displaced 1 DB 1odc A Water 78 Distance 1 1034 Temperature Factor 35 650 A DB 1666 A Water 157 Distance 0 8434 Temperature Factor 24 790 a DB 1som A Water688 Distance 0 9034 Temperature Factor 34 040 A DB 1zgc A Water 60 Distance 1 1504 Temperature Factor 23 210 A DB 1zgc B Water 63 Distance 1 1074 Temperature Factor 19 970 A DB 1vxr A Water 1022 Distance 0 8244 Temperature Factor 29 540 A DB 1gie A Water 1130 Distance 0 8654 Temperature Factor 38 020 A DB 1qif A Water 1164 Distance 0 9254 Temperature Factor 45 910 a DB iqid A Water 1127 Distance 0 9434 Temperature Factor 34 040 A UP 1540 4 pdbtgpk 1 Atom c3 Sybyl Type C 2 Reference Water 643 Temperature Factor 20 340A Conserved 14 Displaced 0 PDB 1h22 A Water 36 Distance 0 4844 Temperature Factor 29 940 A P P PI P P P PDB 1w6r A Water 65 Distance
53. once for each template being provided force allows you to force entry and ignore any warnings or errors Relibase Export Import Commands relibase dump_ligands database lt db radius f gt loat only_ref on off only _ nam es on off Relibase User Guide 189 format mol2 sd f relibase dump_ligands database lt db gt writes out all the ligands in the database lt db gt There are several optional arguments for this command radius float By default the radius is zero which means that only ligands will be written to the output files If set to a larger value in Angstrom all protein residues within this distance of the ligand will also be written to the output files only_ref on writes out all the reference ligands e g if there is gt 1 occurrence of an SOq anion only one ligand will be written out only_ref off writes out all occurrences of ligands etc in the specified database only_names This command provides a listing of reference ligands and corresponding ligand models Used in conjunction with the only_ref on option above this provides a list of unique reference ligands for which images are required Note that this command applies to a single database format Use this command to stipulate which format your ligand file is written out in i e either mol2 or sdf relibase database lt db dump_cavities gt Exports all
54. part of a cavity from this database can be used as query in a similarity search which will find other similar cavities or sub cavities in the remainder of the database Similarity is judged by matching 3D property descriptors pseudocentres that encode the shape and chemical characteristics of each cavity see Pseudocentres Section 1 4 page 98 No sequence information is used which is why the program can detect similar cavities even if they have no obvious secondary structure relationship Visualisation software is provided for displaying the results of cavity similarity searches for comparing query and hit cavities etc see Displaying and Comparing Cavities Section 3 page 101 The CavBase cavity database is closely linked to and may be used alongside Relibase CavBase enables cavity databases to be created from in house protein structures see Building In House Cavity Databases Section 6 page 118 and searched alongside the PDB derived database Uses of CavBase The main uses of CavBase are Inference of function mechanism of active sites by comparing the query cavity with similar cavities of known function mechanism Generation of ideas for novel ligands by observing what is bound to other similar sites Investigation of ligand selectivity and cross reactivity since a ligand known to bind to the query cavity might bind to other similar cavities Identification of novel target sites since the database contains all c
55. running using the Browse Hits Relibase User Guide button in the Results tab or when the search is finished by loading the search via the Stored Search Results pulldown menu in the Stored Results window see Storing Search Results Section 6 1 page 47 6 8 2 Options available on Search Hitlist controls e Restrict search to hitlist named allows you to use a previously saved hitlist Type the name of the required hitlist into the Restrict search to hitlist named box in order to restrict the search to ligands or PDB entries in that hitlist e Save search in hitlist named allows you to save the results of a search in a hitlist type the required hitlist name into the Save search in hitlist named box before you start the search see Storing Combining and Converting Search Results Section 6 page 47 e In the resulting dialog box various options are available e If atoms were selected 3 or more non hydrogen atoms must be selected the dialog box also contains the Superimpose Hits on Selected Atoms check box Click on this if you wish to generate an overlay of the hits see Viewing 3D Substructure Search Results Geometrical Analysis Section 3 5 page 25 Selection of this check box generates a pull down menu from which you can choose to Display matching atoms only Display matching chains or Display entire binding site Once the search has been run the superimposed hits can be viewed on the results page default or loaded into Hermes cl
56. secondary structure browser that permits navigation of secondary structure elements in the protein Relibase User Guide 39 Helices r Helix Vectors r Strands Y Turns METAL GLUA ASN A3 PHE A4 eee SEE gt v Zoom and center on clicking link Turn Display All Turns v Assignment Method Original PDB w e Each cell in the table corresponds to a secondary structure assignment to a given amino acid in the protein Cells contain a condensed description of the secondary structure element in question For turns the convention used is x y sub type for example an inverse gamma turn would be describedas n 3 inverse in the table e Hovering over a cell with the mouse cursor displays the full description of the element in question Turns helices and strands are coloured differently to aid recognition Turns are coloured differently based on type normal are magenta open are cyan and reverse are green and then shaded by length e A given amino acid can be a component in one or more secondary structure elements this particularly applies to turns as single amino acids can be part of several overlapping turns that build up to make a compound secondary structure element e The most common example of such behaviour is well known an ideal a helix in a protein can be viewed as a sequence of 5 residue type turns that overlap with each other Below is an example of an a helix that was assigned using SHAFT starting with normal 4
57. secondary structure elements with a hydrogen bond or a specific Ca Ca distance between the first and the last residue Turns can be up to six residues in length thus a tab exists for each turn length e Within each turn length tab further tabs are available based on the types of turns available for the turn length 2 Residue turns can only be the reverse type 3 Residue can be normal or reverse 4 Residue 5 Residue and 6 Residue turns can be normal open or reverse e To select turn types deactivate the Ignore Turn Type check box to activate and select the allowed options e Each pane allows the user to specify the relative position that a hit residue occupies in the turn Simply deactivate the Ignore Position check box and select from the resultant options e Use the Reset button to return the window to its initial display Relibase User Guide 153 154 Relibase User Guide Relibase User Guide 155 156 Relibase User Guide CHAPTER 6 CREATING IN HOUSE DATABASES Introduction see page 157 Overall workflow see page 157 Ligand templates see page 159 Synonyms file see page 160 Customising the processing requirements see page 161 Structure factors and electron densities see page 163 Processing structures using the web based GUI see page 164 Processing structures using the command line see page 168 SAAN BR WN 1 Introduction e Relibase provides a complete solution for storing and managing both public and prop
58. single turn per chain This does not reflect the full secondary structure assignment in a given PDB entry as a given chain can contain several isolated turns that are not sub components of a helix e A more serious issue with secondary structure classification in the PDB for the purposes of database searching is that the classification method used varies from structure to structure This means that the definition of an N terminus of a helix for example will differ in one PDB entry to the next according to the definition included with the public structure e The secondary structure module in Relibase contains the original PDB assignments of secondary structure but in addition contains a new consistent assignment of turns and helices The turn assignment is based on a machine learning algorithm to cluster various different turn types and was used for a complete assignment of all turns in given proteins This turn assignment was also used to define helices based on multiple turns Furthermore algorithm for the identification of kinks and bends in helices and b strands are implemented 4 6 2 Classification Methodology e A basic description of the methodology used is given here For a more complete description please refer to the following references e Turns revisited A uniform and comprehensive classification of normal open and reverse turn families minimizing unassigned random chain portions O Koch G Klebe Proteins Structure Function
59. the surfaces off and then toggle between the query matched PCs and PCs searched for The same pull down toggle allows you to see quite how much of the initial cavity pdb1bxo 1 has been removed from our tutorial query Unmatched PCs By clicking on other cavities hyperlinked in the Search Results table in the Relibase browser you will be able to compare other less well matched cavity pairs If for example you were to compare any pair of cavities in the worst scoring 170 entries in the table you would be very hard pushed to make any meaningful interpretation 4 4 Further optional analysis What follows is a more detailed analysis of the search we have just carried out The next step of this tutorial assumes you have access to Excel software although clearly there are a number of other pieces of spreadsheet software that allow the import of csv tabulated data and its subsequent manipulation and graphical display 50 e Import the tutorial csv file you saved earlier into Excel or whatever preferred spreadsheet program and check that the Normalised Score column is still in descending order It is a user preference whether or not the Normalised Score rather than the raw Score is used Normalised Score gives some impression of how good a match the hit is as a percentage fit when compared with a perfect match It also gives the impression that you can compare results when using different score functions clearly no more accurately than you cou
60. the Geometric Parameters dialogue box by clicking on the Add 3d button in the Draw window The dialogue box can only be opened when there is a substructure in the white drawing area Select the atoms that are needed to calculate the required parameter by clicking on them with the left hand mouse button click again on an atom to deselect As the number of selected atoms varies the dialogue box will list the parameters that can meaningfully be defined In the example below two atoms have been selected so it is possible to define the distance between them File Ed Options Help Modes Query Sketcher 3d Parameters Click on atom to add to selection F Edit 3D Parameters oo D To construct a parameter object Templates Current selection E he cs Valid parameters Defined paramete Da Distance Define View Controls Valid objects Defined objects Centroid Define ector Define Done LY Hit the appropriate Define button in the list of Valid Parameters e g hit the Define button next to the word Distance to define an interatomic distance atoms do not need to be bonded to one another The defined parameter will be listed in the box labelled 3d Parameters in the top right hand corner of the Draw window In the example below the N C distance has been defined and named D by default Relibase User Guide File Edit iew Atom Bond Options Help Modes Query Sketcher
61. the centre of the bond with the right hand mouse button pick Cyclicity from the resulting pull down menu then select the required option from the second pull down menu Unspecified in this menu means the bond may be either cyclic or acyclic If the bond is already part of a ring the Acyclic option will not be active For bonds which form part of a ring i e cyclic bonds it is also possible to specify the ring size Note When a particular bond is part of more than one ring the smallest ring will be considered when testing the constraint Relibase User Guide e To set a maximum limit on the ring size click on the centre of the bond with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Maximum smallest ring sizes then select the required option from the third pull down menu To set a minimum limit on the ring size click on the centre of the bond with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Minimum smallest ring sizes then select the required option from the third pull down menu e To specify an exact ring size click on the centre of the bond with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Exact smallest ring sizes then select the required option from the third pull down menu e To specify a ring size range click on the centre of the bond with the right hand mouse button pick Cyclicity from the resu
62. the fifth cavity in the list occupied by a rhodamine 6G ligand viz CAV pdb1jus 5 32 Relibase User Guide Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Relibase Hits 1 15 of 15 pdb1jto pdb1jt pdbijtx pdb1jty pdbijum pdbijup pdbiqvt pdbiqvu pdbirkw pdbirpw pdb2dtz pdb2g0e pdb2gby pdb2hq5 Hits 1 15 of 15 15 hits for query QacR Export XML Hitlist Save in Hitlist Save Search Results Browse Hit Headers PDB Entry Code Keyword Search Results reiibase 3 0 0 res Cavities in PDB Entry 1jus a Cavity kome Ligand Ligand a g g pdbtjus 1 338 2 pdbijus 2 353 1 pdb1jus 3 pdb tjus 4 1855 5 pdbtjus 6 Warning Cavities and are un 2932 8 suitable larger than 30004 may be slow to load in the visualiser for use as a reference cavity when searching pdb1jus 6 3156 6 e Click on the CAV pdbIjus 5 link This takes us to the cavity information page for this cavity Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Sketcher Hitists Stored Results Help Hits 1 16 of 16 pdb1jto pdb1jt pdb1jtx pdb1jty pdbijum pdbijum pdbijup pdbiqvt pdbiqvu pdbirkw pdbirpw pdb2dtz pdb2gde pdb2gby pdb2hg5 Hits 1 16 of 16 Cavity pdb1jus 5 in PDB Entry 1jus Keyword Search Results reibase 3 0 0 Volume of cavity A gt 2932 7
63. to be a turn The subdivision nominally could results in 15 subset namely normal inverse and open turns of 2 3 4 5 and 6 residues respectively In practice 3 normal turn families 4 open turn families and 5 reverse turn families were observed and clustered e For each turn back bone torsion angles were evaluated and used as coefficients of an N dimensional vector Where N was the number of backbone torsion angles in each turn type The vectors were then used for clustering based on Euclidian distance by making each observed sequence vector a node in an Emergent Self Organising Map ESOM The ESOM clusters similar vectors into similar regions leading to a self organised classification of turns for structures in the training set e Analysis of the results led to identification of 158 turn types each of which is identified in Relibase Using the trained ESOMs the turn types can then be assigned programatically to all entries in the PDB leading to a consistent turn type assignment across all entries 4 6 4 Turn Types e The resulting classification of secondary structure enumerates many different turn types Each have been given a classification based on residue length turn type open reverse or normal 36 Relibase User Guide and a sub classification based on the cluster occupied by a given turn type These assignment names are based on the ranges of internal backbone angles within a give turn type There is no firm nomenclatu
64. to convert file formats e OpenEye Toolkit e Daylight Toolkit Note that the latter two options are experimental and have not been extensively tested in house e To configure the ligand diagram generation go to the Help page and click on the Ligand Diagram Generation Configuration hyperlink This will take you to the Ligand Diagram Generation Configuration page Relibase PDB Entry Code fve Home Text Search Sequence Search SMILES Search Sketcher Hitiists Stored Results Help Ligand Diagram Generation Configuration reibase 3 1 0 Options View Current Settings Test Diagram Generation Configure With Manfred Hendiich 1994 1999 and Cambvidge Cryatatlographic Data Centre 1999 2014 WS vac egy e Select the package that you want to use for your ligand diagram generation on the left hand side and enter the information required e Tripos SYBYL path to the SYBYL installation directory TA_3DB and the location of the licence file or the port hostnmae of the licence server TA_LICENCE_FILB e ChemAxon MarvinBeans the location of the MarvinBeans converter MARVINBEANS_PATH and the location of the Babel executable BABEL_PATH e OpenEye Toolkit the location of the mol2gif converter MOL2GIF_PATH and the location of the oe_license txt file OE_LICENSE e Daylight Toolkit the path to the Daylight installation directory DY_DIR and the location of the Daylight licence file D
65. water molecule is potentially an error Criteria for Notification Low B factor 13 780 average 34 921 Octahedral coordination RMS 0 1404 Short bonds lt 09A Relibase User Guide 9 5 7 5 8 Cavity Information All cavities in a protein structure are listed with their volumes and any ligands they contain Note Very large cavities gt 3000 are usually of little interest they are often ill defined gaps between large protein domains Cavity information for any database entry can be accessed by clicking on the Cavity Information button at the bottom of either Protein or Ligand Information pages see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 Secondary Structure Information Details of helices beta sheets and turns in the protein can be viewed and displayed in 3D via the Secondary Structure Information button at the bottom of each Protein Information page see Secondary Structure Information Section 4 6 page 35 6 A Typical Relibase Entry Embedded 3D visualisation via Astex Viewer v Packing LJ _ Schematic Bibliographic and chemical text information 10 Relibase User Guide SERINE PROTEASE X RAY STRUCTURE OF THE COMPLEX OF HUMAN ALPHA THROMBIN WITH THE INHIBITOR SDZ 229 357 Compound MOL_ID 1 MOLECULE ALPHA THROMBIN CHAIN A B F E EC 3 4 21 5 Reference J MED CHEM 41 1998 3664 JKALLEN MOL_ID 1 ORGANISM_SCIENTIFIC HOMO SAPIENS ORGANIS
66. with the Show button Click on the relevant Show hyperlink under the column headed Shown in Hermes to view the water mediated protein ligand contacts in Hermes Relibase User Guide 33 4 5 34 Cavity Information Cavity information for any database entry can be accessed by clicking on the Cavity Information button at the bottom of either the protein entry or ligand pages Secondary Structure Secondary Structure Information Waters Water Information Cavities Cavity Information If you are on a ligand page and there is information available for the ligand cavity clicking on this button takes you to a page displaying the volume of the cavity containing the ligand header information and the ligand chemical diagram if available Also the visualiser see Displaying and Comparing Cavities Section 3 page 101 will open with the selected cavity loaded If you are on a protein entry page clicking on the Cavity Information button takes you to a page listing all the cavities in the protein structure sorted in ascending order of cavity size and any ligands they contain clicking on a ligand diagram links you to the Ligand Information page Cavities in PDB Entry 8aat Cavity pdbSaat 1 pdbBaat 2 pdb aat 3 pdbSaat 4 pdbBaat 5 pdb aat 6 pdbSaat 7 Warning Cavities larger than 30004 many be slow to load in the visualiser and are unsuitable for cavity searching pdbSaat 6 3387 2 pdbBaat 9 3541 2 Similarity Sea
67. within a distance threshold of 1 0A the points are simply counted In 2 and 3 this simple distance function on off is replaced by a more complicated more accurate block function in order to better evaluate the degree of overlap between two surface patches The surface points on each patch created by the pseudocenters are not only counted but also weighed according to the closest distance between two surface points two surface points that are close give a higher contribution to the score The difference in 2 and 3 is in the distance that is used in the block function in scoring function 2 the distance is squared leading to a different distance dependence of the score Scoring function 3 also has a better pair selection of pseudocenters improving the calculation of overlap At present there is insufficient information regarding the relative merits of these functions all appear to work reasonably well The functions are on different scales so comparisons between the values from different functions are invalid However for any given function a higher number always suggests closer similarity 2 Accessing Cavity Information for Relibase Database Entries e Cavity information for any database entry can be accessed by clicking on the Cavity Information button at the bottom of either Protein or Ligand Information pages WaterBase Water Information CavBase Cavity Information Relibase User Guide 99 e Ifyou are on a Ligand Informa
68. you wish to add or alter this path post installation you will need to manually edit your SRELIBASE_ROOT bin relibase setup sh sh or bash shells and SRELIBASE_ROOT bin relibase setup tsch shell files For the former sh or bash shells add the line SCCP4_MASTER setup scripts sh ccp4 setup to the end of the relibase setup sh file note that CCP4_MASTER should be the top level of your CCP4 software installation For example local ccp4 setup scripts sh ccp4 setup For the latter tsch shell add the line source SCCP4_MASTER setup scripts csh ccp4 setup to the end of the relibase setup file For example source local ccp4 setup scripts csh ccp4 setup Structure factors for in house structures can be stored in Relibase For structures in the core reli database structure factors can be fetched on a per entry basis from the Uppsala Electron Density Server EDS hitp eds bmc uu se eds if available Downloaded MTZ files are cached in SRELIBASE_ROOT relibase_htdocs tmp EDS_MTZ To disable caching of EDS MTZ files set the DISABLE_EDS_CACHE parameter to 1 0 to enable it in the SRELIBASE_ROOT bin relibase setup config file To disable access to the EDS server completely set the DISABLE_EDS_ACCESS parameter to 1 0 to enable it in the SRELIBASE_ROOT bin relibase setup config file e If you are
69. you wish to monitor The maximum distance in a distance constraint for instance is set at a default of only 3 5 Run the search see Running a Search Section 6 8 page 74 Hints for 3D Substructure Searching and Tabulating Geometries To learn how to run searches involving protein and or water molecules read the section on nonbonded interactions see Non bonded interaction searching Section 6 6 page 72 Geometric parameters are often defined so that they can be analysed later using histograms All required geometric parameters must be defined in the drawing window when the substructure is drawn They cannot be defined after a search has been run Think carefully about the problem being studied to ensure that you have specified geometric parameters that adequately describe that problem The obvious choice is sometimes not the best Once defined any geometric parameter can be used as a search constraint by specifying suitable limiting values In setting geometric constraints it is often useful to survey typical values found in the Relibase database before deciding the limiting values to be used in a subsequent search If you have drawn a complicated query e g with multiple distance constraints the search may be slow To check whether the hits you retrieve are of the desired type you can interrupt the search after it has found a few hits This enables you to inspect the hits found so far Non bonded interaction searching A hi
70. 0 9374 Temperature Factor 30 630 A PI P P PI H DB 1h23 A Water 44 Distance 04164 Temperature Factor 31 440 A 9 4 8 The Analysis Table Clashes with Proteins e This data is not presented in the analysis table as it is first calculated Click on the relevant checkbox in the Ligand binding site area at the base of the table and then click on the Recalculate Table button Each number in the column represents the number of atoms in the reference ligand which clash with atoms in the relevant superimposed chain A clash is defined as being an atom atom distance of less than the sum of the Van der Waal s radii by 0 1A or more e Each numeric entry in the column links to a table giving further information about the clashes found for that chain Relibase User Guide 95 96 ConservediDisplaced Waters Reference chain PDB 1acj Reference ligand THA_999_pdb1acj_1 Key Grey conserved Blue displaced Reference Water 634 Temperature Factor 24 0604 Conserved 10 Displaced 1 PDB 1odc A Water 78 Distance 1 1034 Temperature Factor 35 650 A PDB 1e66 A Water 157 Distance 0 8434 Temperature Factor 24 790 a PDB 1som A Water 688 Distance 0 9034 Temperature Factor 34 040 A PDB 1zgc A Water 60 Distance 1 150A Temperature Factor 23 210 A PDB 1zgc B Water 63 Distance 1 1074 Temperature Factor 19 970 A PDB 1w6r A Water 65 Distance 0 937 Temperature Factor 30 630 A PDB 1vxr A
71. 3 13 2 e 0 492 HYDROLASE INHIBITED BY TABUN CRYSTAL STRUCTURE OF MOUSE pdb2h9y 13 13 2 0 791 18 6 HYDROLASE ACETYLCHOLINESTERASE COMPLEXED WITH M N N N TRIMETHYLAMMONIO TRIFLUOROACETOPHENONE CRYSTAL STRUCTURE OF MUTANT 5203A OF pdb2ha5 12 5 8 13 2 0 756 18 6 HYDROLASE ACETYLCHOLINESTERASE COMPLEXED WITH ACETYLTHIOCHOLINE A MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX pdb2jez 8 5 7 13 0 1 006 HYDROLASE WITH TABUN AND HLO 7 CRYSTAL STRUCTURE OF THE MOUSE pdh ineri ta aA 128 os 0 990 HERO ACETYLCHOLINESTERASE GALLAMINE COMPLEX i MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX pdb2jey 13 5 6 12 7 0 619 18 6 HYDROLASE WITH HLO MUS MUSCULUS ACETYLCHOLINESTERASE IN COMPLEX pdb2jf0 9 5 5 12 6 1 979 HYDROLASE WITH TABUN AND ORTHO 7 F CRYSTAL STRUCTURE OF MOUSE 3 787 18 HYDR ab 106 12 23 12 s om we DROLASE ACETYLCHOLINESTERASE IN THE APO FORM CRYSTAL STRUCTURE OF MOUSE ACETYLCHOLINESTERASE INHIBITED BY NON AGED FENAMIPHOS CRYSTAL STRUCTURE OF THE MOUSE ACETYLCHOLINESTERASE PROPIDIUM COMPLEX pdb2jgf 12 54 12 3 6 0 650 Ea HYDROLA pdbin r 12 54 12 2 6 0 654 5 HYDROLA Browse the next 15 hits 3 5 Viewing a Hit and Comparing It with the Query e To view this hit click on the pdb2 f0 9 link This will display a browser page giving details of the hit You will see that the hit is an acetylcholinesterase AChE structure Relibase User Guide 37 Comparison of cavities pdb1jus 5 and pdb2jf0 9
72. 380 1 315 1 920 0 656 0 533 0 574 0 521 0 517 0 522 0 667 0 734 0 755 0 648 0 883 0 916 0 987 N GSG 0 587 0 556 0 620 0 620 0 531 0 511 0 547 0 537 0 500 0 616 0 572 0 504 0 966 naa naa 0 768 0 682 0 708 0 519 123 ossa 0 725 osno fosa n 772 n AATF Nn EEA n ARA 9 4 4 The Analysis Table Sidechain Movements e The fourth column gives information on the number of significant sidechain movements if any with respect to the reference chain first figure The movement is measured between the centroids calculated for all the heavy atoms within the sidechain for both reference and superimposed chains Also given are the number of sidechain torsion angles that differ significantly from those in the reference chain second figure The default threshold for what constitutes a significant atom movement is 1 0A The default threshold for what constitutes a torsion change is 10 degrees These thresholds can be changed by altering the relevant figures in the Sidechain movements and Torsion angle changes box in the Protein flexibility area at the base of the table and then clicking Recalculate Table The column can also be hid from view by clicking off the appropriate check box in the Protein flexibility area prior to recalculation e Each numeric entry in the Sidechain Movements column links to an expanded list of residues involved in movement and the distance of sidec
73. 3d Parameters Click on atom to add to selection Edit 3D Parameters n DA To construct a parameter object Templates Current selection i m a Valid parameters Options View Controls Valid objects Defined objects e Once a parameter has been defined its value can be constrained see Applying Constraints Section 12 page 144 e You can continue to define other parameters Once all parameters of interest have been defined hit Done to close the Geometric Parameters dialogue box 11 3 Defining Geometric Parameters Involving Objects e The procedure is exactly the same as for parameters involving only atoms see Defining Geometric Parameters Involving Atoms Section 11 2 page 140 except that objects are picked from the Defined Objects list e For example to specify the distance between a centroid and an atom first create the centroid see Defining Geometric Objects Section 10 2 page 138 Relibase User Guide 14 File Edit View Atom Bond Options Help Query Sketcher 3d Parameters gt Valid parameters Defined paramete __ Options Valid objects Defined objects M Options Delete c u o n s p F ci any omer Then select the atom by clicking on it and the centroid by clicking on its object name in the list of Defined Objects CENTI in the above example File
74. 5 Header Compound TRANSCRIPTION CRYSTAL STRUCTURE OF THE MULTIDRUG BINDING TRANSCRIPTIONAL REPRESSOR QACR BOUND TO RHODAMINE 6G MOL_ID 1 MOLECULE HYPOTHETICAL TRANSCRIPTIONAL REGULATOR IN QACA S REGION CHAIN B D A E SYNONYM QACR REPRESSOR ORF 188 ENGINEERED YES MUTATION YES Ligands Reload Go to Table of Previous Cavity Similarity Search Results Click here to reload Cavity e The cavity will be displayed in 3D within Hermes The Cavity Controls window will also come up Go to the Search Setup pane of the viewer by hitting the right hand tab within this window Relibase User Guide 33 Cavity Controls You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1jus 5 in reli Hit None n Display Controls Search Setup Use any of the methods below to select pseudocentres PCs in the Query cavity to be searched for Jeftolicking on PCs righclick on objects to pick nearby PCs clicking on the lick boxes below Use the pulldown options below to sort the pseudocentres by different criteria 1st Sort Backbone or Sidechain 2nd Sort Pseudocentre Type v 3rd Sort Residue Type v Column 1 PseudoCentres a O Backbone a O Acceptor a oO Donor a O Pi a O Sidechain Metal a O Acceptor O Aliphatic a O Aromatic a O Donor O DonorAcceptor a O Pi To begin with all
75. 5 06 28 ASCII XML tmp_1u4d adf_rb3 Protein 3 Public Not owner Tue Aug 12 2008 13 51 47 ASCII XML tmp_cav_1ud4 adf_rb3 Cavity 14 Public Not owner Wed Aug 13 2008 15 06 55 ASCII XML Cavity Set esterase_cav esterase henderson Protein 683 Public Change Delete Mon Aug 18 2008 15 02 12 ASCII XML hydrolase henderson Protein 10591 Private Change Delete Mon Aug 18 2008 15 50 20 ASCII XML Make New Set nad_lig henderson Ligand 1611 Private Change Delete Thu Aug 21 2008 11 36 25 ASCII XML Submit nad_lig2 henderson Ligand 4 Private Change Delete Thu Aug 21 2008 11 46 42 ASCII XML Fpdb207v2 I pdb2c58 7 lig_suc henderson Ligand 566 Private Change Delete Thu Aug 21 2008 11 49 08 ASCII XML keyword _kinase adf_rb3 Protein 2747 Public Not owner Tue Aug 26 2008 10 11 37 ASCII XML M Babac8a 9 T pab2jto 4 short_rand_prot adf_rb3 Protein 3 Public Not owner Tue Aug 26 2008 10 21 45 ASCII XML Fpabtei9 1 I pdb2ckm 1 key_ oxidoreductase adf_rb3 Protein 4655 Public Not owner Tue Aug 26 2008 13 27 21 ASCII XML I pabtyas 9 I pdb2aox 5 bode henderson Protein 173 Private Change Delete Wed Aug 27 2008 14 38 40 ASCII XML henderson Cavity 3506 Public Change Delete Wed Aug 27 2008 14 43 23 ASCII XML M pdb2gfb 5 M pdbicie 2 I pdb2dgz4 I pdb2c p 11 I pab2f0x 11 I pdb3bes 12 I pdbigpk 2 M pdb2c58 6 I pdb2c5g 3 M pdb1mx9 7 Hitlist Operations for User henderson Ligand Set 1 Operation Ligand Set 2 New Set Gladatawe Dsanieas Select existing hit
76. AB Click a histogram slice to view its entries and constraint matches Histogram Label T1 Torsion Distribution Slice Size 10 Update Torsion Angle Distribution 32 15 15 13 i 10 A 9 6 180 170 160 150 140 130 120 110 100 90 60 70 60 50 40 30 20 10 0 10 20 3040 50 60 70 80 90 100 110 120 130 140 150 160 170 Total Distribution Size 157 e By default histograms for angles and torsion angles are binned at 10 degree intervals those for distances at 0 2A intervals To alter the distribution bin size enter a distribution slice size and hit Update The histogram will be updated to reflect the specified bin size The number of observations in each interval is shown at the top of each bar Click on the individual bars to load all hits that make up that bar into the Relibase ligand browser see Using the Ligand Browser Section 3 3 page 22 e Specified parameter values for hit structures can be saved to a file for later analysis Choose from e Export Histogram Data as CSV outputs the current histogram in CSV file format Export Histogram Data as TAB outputs the current histogram in tab format suitable for input to Vista the statistical analysis package distributed with the Cambridge Structural Database System http www ccdc cam ac uk products csd_system vista 3 5 2 Viewing 3D Superposition of Hits e When you have asked for query atoms or centroids to be superimposed see Running a S
77. About This User Guide This user guide is a practical guide to using the Relibase and Relibase tools for searching protein ligand structures It includes instructions on using the graphical user interface Hermes for Relibase as well as providing help on relevant scientific issues Use the lt and gt navigational buttons above to move between pages of the user guide and the TOC and Index buttons to access the full table of contents and index Additional on line Relibase resources can be accessed by clicking on the links on the right hand side of any page An extensive set of tutorials are also available for Relibase Tutorials can be accessed by clicking on the Tutorials link on the right hand side of any page The Relibase user guide is divided into the following sections CHAPTER 1 THE RELIBASE DATABASE see page 2 CHAPTER 2 GENERAL FEATURES OF RELIBASE see page 17 CHAPTER 3 RUNNING RELIBASE SEARCHES see page 57 CHAPTER 4 RUNNING SIMILAR CAVITY SEARCHES see page 97 CHAPTER 5 USING THE RELIBASE SKETCHER see page 119 CHAPTER 6 CREATING IN HOUSE DATABASES see page 157 Relibase User Guide 1 CHAPTER 1 THE RELIBASE DATABASE Noa bk WN 2 Coverage of the Relibase Database see page 2 Database Entries see page 2 Entry codes see page 2 Database Statistics see page 3 Database Information content see page 3 A Typical Relibase Entry see page 10 Coverage of the Relibase Database The datab
78. C PDB 1cfj A 0 419 None 144 None None 2 c oe 0 412 None 1 6 None 49 36 2 c ong Je34 oa 2 1 3 None 48 26 2 c Search results can be viewed in Astex Viewer or if the Automatic Visualiser Updates tick box is activated Hermes will automatically come up when a superposition is completed It is possible to download mol files of all the structures in their superimposed frame of reference by hitting the Download Superimposed structures link Ligands protein chains and waters are all downloaded The current reference ligand and current reference chain are displayed above the table If you want to recalculate the superposition with a different reference chain this can be done by clicking on the check box next to the desired reference chain and pressing the Change reference chain button All the values in the table will be recalculated accordingly There are several headers in the analysis table e Protein Chain see The Analysis Table Protein Chain Section 9 4 1 page 91 e RMS see The Analysis Table RMS Section 9 4 2 page 91 e C Alpha Movements see The Analysis Table C Alpha Movements Section 9 4 3 page 91 e Sidechain Movements see The Analysis Table Sidechain Movements Section 9 4 4 page 92 e Mutations and Insertions see The Analysis Table Mutations and Insertions Section 9 4 5 page 93 Relibase User Guide e Ligand Overlap see The Analysis Table Ligand Overlap Section 9 4 6 page 94 e Conserved Waters see The A
79. Controls ee aR i Mi Cals VAN E 7 Wi 0 ia gt IF K Options B e ne Delet l hal START SEARCH c H O N S P F Cl Any Other N Ligand M Bond Single Relibase User Guide To specify that the N atoms are only connected to one non hydrogen atom right click on the first N atom and select Number of connections to non hydrogen atoms 1 from the pull down then do similarly for the second N atom Click on the Search button This will take you to the Start Search window where a number of filters and other search options can be defined including the search name Select the Hitlist Controls tab and type amidino into the Save search in hitlist named box By default only 1000 hits will be returned for this search To return all substructure matches select the Hit Limits tab and select the Show all hits radio button Hit the Start button to initiate the search the amidino hitlist saved from the SMILES string search will be overwritten by default Relevant hits are displayed in the Results window during the search A new browser window containing all the hits is launched when the search has completed Return to the sketcher by selecting the Query Sketcher tab Delete the amidino fragment by clicking on Edit and Delete All Ensure the molecule type is set to Ligand and select the Naphthalene template by clicking on the Other button in the Templates part of the interface L
80. D Sketcher retibase 2 1 0 Atom Bond Help Query Sketcher 3d Parameters Click on atom to select click and drag to select several atoms 2 e Before closing the Add 3d window select the distance D and click on the Options button This launches a dialogue box which will allow you to alter the limits on the distance constraint Change the upper limit to 2 0 then click OK and then Done to finish the definition e Click Search e Inthe Start Search window specify that the search is to be restricted to X Ray Structures only with a Lowest resolution of 2 0 Angstrom then click Start to initiate the search e Click on any of the entries that appear in the Results tab to view hits generated for this search Results will be presented in a second browser window while the search is still running e Once the search is complete look at a hit such as PDB entry Icv2 which is a very nice example of the occurrence of pi pi stacking interactions Relibase User Guide 29 30 Relibase User Guide 3 Tutorial 3 An Introduction to the Cavity Information Module and Hermes 3 1Overview 3 1 1 Objectives To find ligands that might bind to a particular protein binding site The binding site of interest will be used as the query cavity in a CavBase search looking only for hit cavities that contain ligands If a hit cavity is sufficiently similar to the query then the ligand occupying that hit cavity might also bind to the query cavity 3 1 2
81. Descriptors Help Highlighting v Depth Cueing Stereo ShowH HideH ShowUnk Hide Unk Graphics Objects Style Wireframe v Picking Mode Select Atoms Clear Measurements x x y y z z x 90 x 90 y 30 y 90 2 90 z 90 amp gt 4 T zoom zoom Atom selections y Display Movable I V Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1f0r 2 in reli Hit CavBase Hit pdb1c51 3 in reli Display Controls Search Setup Show Pseudocentres Query Matched PCs v Hit Matched PCs v Link matching PCs with lines Relibase User Guide 103 3 3 Customising the Cavity Display 3 3 1 Using the Graphics Object Explorer to Control the Display of Cavity Objects 104 When a cavity is opened two dockable windows also become visible to the left of the Hermes screen The upper is the Protein Explorer Window which can be used to control many aspects of structure display please refer to the Hermes documentation for further information The lower is the Graphics Object Explorer which is used to control the display of graphical objects which are not chemical structures The display of cavity surfaces and pseudocentres is controlled by this window Hierarchical trees are used to list the objects molecules surfaces pseudocentres etc that are currently loaded Each cavity
82. Edit View Atom Bond Options Help Query Sketcher 3d Parameters E Edit 3D Parameters To construct a parameter object Select atoms in main window Define parameter or object ENTI 04 l Valid parameters Defined paramete Distance Define Other Options View Controls Valid objects Defined objects Centroid ector Templates C H O NS P F Cl Any Other 142 Relibase User Guide Then hit the relevant Define button next to the word Distance in the above example File lit i N d Options Help 3d Parameters Modes Query Sketcher Click on atom to add to selection F Edit 3D Parameters o Bd To construct a parameter object Current selection Valid parameters Defined parametei Templates Options View Controls Delete Valid objects Defined objects CENT1 gm e Once a parameter has been defined its value can be constrained see Applying Constraints Section 12 page 144 e You can continue to define other parameters Once all parameters of interest have been defined hit Done to close the Geometric Parameters dialogue box 11 4 Renaming Geometric Parameters e By default distances are named D1 D2 etc angles A1 A2 etc torsions T7 T2 etc e To rename a parameter select it in the 3D Parameters list top right hand corner of Draw window then hit the Options but
83. Fe Relibase User Guide 73 6 7 6 8 74 atom and ligand O atoms e In cases where the O is drawn first if the search returns two O groups in the same ligand that coordinate the Fe only one hit would be added to the hitlist i e only the first ligand hit if the search returns 2 different ligands that contain O atoms coordinating the Fe two hits would be added to the hitlist i e one hitlist entry for each ligand e If the Fe atom is drawn first and the search returns two different ligands that contain an O atom coordinating the Fe one hit is returned in the hitlist i e only one contact to a given Fe atom is added to the hitlist Drawing a 2D 3D Substructure For instructions on substructure drawing refer to the section on using the Relibase sketcher see CHAPTER 5 USING THE RELIBASE SKETCHER page 119 For information on setting up 3d constraints please refer to the relevant section in the chapter on using the Relibase Sketcher see Applying Constraints Section 12 page 144 Running a Search Having created a 2D or 3D substructure query in the Sketcher click on the search button on the left hand side of the Sketcher window The Start search dialogue box will come up Hitting the Start button initiates the search As the search progresses hits are displayed in a new Sketcher Results pane In addition any 3D parameters are also displayed Clicking on any line in the Results pane links through to the appropriate Ligand p
84. Information Cavity Information Secondary Secondary Structure Information Structure e The different sequences can be displayed by clicking on the hyperlink for the chain of interest e g pdbla0I1 A above e Clicking on the sequence hyperlink launches the Protein Chain Sequence page with a sequence display and under this a sequence search form e Forms of the type shown below are the starting point for a similar chain search using one chain in the entry as a reference Protein Chain pdb1a01 A YVLSPADKTNVKAAWGKYGAHAGEYGAEALERMF LSF PTTRTYF PHF DLSHGSAQVKGHGRRVADAL TNAVAHYDDMPNAL SALSDLHAHKLRVDPVNF RLLSHCLLVTLAAHL PAEFTPAVHASLDRF LASVSTVLTSRKYR Search for Similar Chains Minimum Sequence Identity Maximum Sequence Identity Use Databases Select existing hitlist Use Hitlist Save in Hitlist I Overwrite Existing Hitlist M Show Ligands Submit e Minimum Sequence Identity and Maximum Sequence Identity boxes can be used to specify the required sequence identity as a percentage with respect to the reference chain default is 100 Use Databases allows you to select which database or combination of databases is searched The default setting is to search All databases Use Hitlist allows you to to speed up the search by restricting it to a previously identified list Relibase User Guide which can be selected from the pop up menu next to Use Hitlist The default is Select existing hitlist until a hitlist is s
85. M_COMMON HUMAN X RAY DIFFRACTION Cell a 71 000 b 72 200 c 73 100 alpha 90 000 beta 100 700 gamma 90 000 Spacegroup C121 2 300 Resolution Unavailable 10 6 1998 Deposition Date 2D diagrams of ligands Ligands DFE in PDB Entry 1bhx Water Mediated Protein Ligand Contact Paths 3 R56 in PDB Entry 1bhx Water Mediated Protein Ligand Contact Paths 3 Sequence data which can be used in a Similar Chain Search Protein Chain pdb1bhx A SGEADCGLRPLFEKKSLEDKTERELLESYI Comprehensive 3D visualisation and exploration via Hermes the protein structure of Lhiv is shown below Relibase User Guide 11 3D structure of binding site Lhiv 12 Relibase User Guide Crystal packing of protein ligand binding site 1qs4 Relibase User Guide 13 Information on water structure and water mediated protein ligand contacts General Information on Water Structure in Entry pdb1a3e 1 206 Water models Water molecules Water molecules per residue 0741 Average B factor 34 921 AZ Standard deviation of B factors 10 231 Dubious Water Molecule Criteria for Notification 13 Low B factor Octahedral coordination RMS 0 1404 Short bonds lt 09A 13 780 average 34 921 Cavity information 14 Relibase User Guide Information on secondary structure Relibase User Guide Cavities in PDB Entry Saat Warning
86. Relibase User Guide 5 2 Hermes Hermes is a program for visualising protein structures in three dimensions with particular emphasis on functionality for the analysis of protein ligand binding interactions Hermes can be launched from any protein entry or ligand page by hitting the Show in Hermes button within the Hermes Controller section of the interface Hermes Controller Show in Hermes Automatic Visualiser Updates To have the visualiser update automatically to display the structure currently shown in the browser switch on the Automatic Visualiser Updates check box If this is switched off the current structure will remain in the display until the Show in Hermes button is clicked again Use of Hermes is covered in detail elsewhere follow the Hermes documentation link on the right of this page 6 Storing Combining and Converting Search Results 6 1 Search results can be saved in one of two ways depending on which type of search has been run e Storing of search results see Storing Search Results Section 6 1 page 47 e Saving of search results in hitlists Hitlists are lists of entries saved from Relibase searches stored separately for each Relibase user Relibase uses three types of hitlist protein ligand and cavity Storing Search Results Results from text searches similar binding site superpositions sequence searches and cavity similarity searches can be stored This is done by typing a search name into th
87. S Handed Helices Allow Alpha Hel C Allow Gamma Helix 7 Allow Omega Hel Other Must not be in a heli Helix Length Minimum Length oH Maximum Length 1 ooo Reset Reset Constraints 1 f Helices Sheets amp Strands Turns Ok Cancel 13 Defining Secondary Structure Elements 13 1 Overview e Secondary structure elements can be defined in 2D and 3D sketcher searches This can be combined with other search tools to provide powerful restricted searches of protein amino acids in given loop conformations 146 Relibase User Guide e Secondary structure assignments can be accessed via protein and ligand information pages and viewed in Astex Viewer see Viewing Secondary Structure Assignments Section 4 6 6 page 38 e An overview of the methodology involved in compiling the secondary structure module is provided elsewhere see Secondary Structure Information Section 4 6 page 35 13 2 Constraining a Protein Residue to be in a Particular Secondary Structure Element e After sketching an amino acid in the sketcher see Using Substructure Templates Section 6 page 133 right click on any atom in the amino acid and select Secondary Structure Constraints from the resultant pull down menu This will launch a Secondary Structure Constraints dialogue window Secondary Structure Constraints Helices Sheets amp Strands Turns Helix Properties Terminu
88. Set element type 4 4 Addition of Hydrogen Atoms e Hydrogen atoms may be drawn in the same way as any other type of atom or they may be defined implicitly It is only possible to add implicit hydrogens to carbon atoms since the number of hydrogens on heteroatoms cannot be safely inferred Note if using protein and ligand templates H atoms are already defined implicitly on the templates themselves see Using Substructure Templates Section 6 page 133 e To add hydrogen atoms implicitly to carbon enter Draw or Select mode click on an atom with the right hand mouse button pick Number of Hydrogens on Carbon from the resulting pull down menu then select the number of hydrogens required from the second pull down menu Unspecified in this menu means that any number of hydrogens is allowed Selecting Generate automatically adds the appropriate number of hydrogen atoms to the carbon atoms so as to satisfy the valency requirements picking Clear removes them e Hydrogen atoms can also be added to any carbon atom already drawn on the sketcher canvas by selecting the Hydrogen Generation option from the Options top level menu Whilst this option is in effect hydrogen atoms are also automatically added to all new carbon substructures as they are added onto the Sketcher canvas Note also that removal of hydrogens cannot be carried out Clear whilst the Hydrogen Generation option is on e Hydrogen atoms may also be drawn explicitly in the same way as any
89. TSKYR LRVDP VNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR This alignment has been carried out using FASTA 3 5 Sept 2006 optimized e Conserved residues are coloured blue residues that are similar are coloured red and residues that are completely different are coloured black e To compare the reference chain to another specific protein chain use the Protein Chain Alignment option Relibase User Guide 83 Protein Chain Alignment PDB chain identifier Align e Enter the PDB chain identifier i e 1a01 B and hit Align The sequence identity is presented for the two selected chains and the two complete chains are aligned As before conserved residues are coloured blue residues that are similar are coloured red and residues that are completely different are coloured black Note the PDB code part of the chain e g 1a01 is not case sensitive however the identifier e g A is case sensitive If an entry has gt 26 chains it is assigned an uppercase chain identifier A B etc however if the entry has lt 26 chains it is assigned a lowercase chain identifier a b 8 1 Hints for Similar Chain Searching e Similar chain searching is recommended for retrieving a complete list of structures for a protein For example a sequence search for thrombin will ensure that only thrombin is retrieved as a hit whereas a keyword search will retrieve many other proteins that are linked to thrombin either in structure or in biochemical fu
90. WANAGA D TYPKE R HUBER 2 W BODE HYDROLASE METALLOPROTE INASE 21 APR 93 1AST ASTACIN E C 3 4 24 21 THE EUROPEAN FRESH WATER CRAYFISH ASTACUS ASTACUS L W BODE F X GOMIS RUETH W STOECKER METALLOENDOPEPTIDASE 26 MAY 95 1ATL MOL_ID 1 2 MOLECULE ATROLYSIN C 3 CHAIN A B C D 4 SYNONYM HEMORRHAGIC TOXIN C FORM D 5 EC 3 4 24 42 6 HETEROGEN SC 44463 MOL_ID 1 2 ORGANISM_SCIENTIPIC CROTALUS ATROX 3 ORGANISM_COMMON WESTERN DIAMONDBACK RATTLESNAKE 4 OTHER_DETAILS ISOLATED FROM VENOM D ZHANG 1 BOTOS F X GOMIS RUETH R DOLL C BLOOD F G NJOROGE 2 J W FOX W BODE E F MEYER COMPLEX SERINE PROTEASE INHIBITOR 12 SEP 97 1aus HUMAN CATHEPSIN G AST 2 iaAsT 3 iasT 4 1AST 5 1ATL 2 1ATL 3 1ATL 4 1ATL 5 1ATL 6 1ATL 7 1ATL 8 1ATL 9 1ATL 10 IATL 11 IATL 12 iATL 14 1ATL 15 To save the hitlist of protein entry codes as an XML format file click on Export XML Hitlist In the resulting pop up window enter a name for the exported hitlist then click OK Note any saved XML hitlist can be read back into Relibase see Loading XML Format Hitlists Section 6 8 page 55 To save the hitlist of protein entry codes on the Relibase server click on the Save in Hitlist hyperlink In the resulting pop up window enter a name for the hitlist then click OK Using the Ligand Browser Search results for ligand based searches e g ligand name ligand entry code Smiles and Sketcher searches see Starting
91. Water 1022 Distance 0 824A Temperature Factor 29 540 A PDB 1gie A Water 1130 Distance 0 6654 Temperature Factor 38 020 2 PDB 1qif A Water 1164 Distance 0 9254 Temperature Factor 45 910 A PDB 1gid A Water 1127 Distance 0 9434 Temperature Factor 34 040 a HUP 1540 A pdbigpk 1 Atom c3 Sybyl Type C2 Reference Water 643 Temperature Factor 20 3404 Conserved 14 Displaced 0 PDB 1h22 A Water 36 Distance 0484A Temperature Factor 29 940 A PDB 1h23 A Water 44 Distance 0 4164 Temperature Factor 31 440 A e Clicking on the header to the column links to a concatenation of the individual clash data for each relevant chain All chains are represented Relibase User Guide CHAPTER 4 RUNNING SIMILAR CAVITY SEARCHES Noa kh WN 1 1 1 2 Introduction and Background Theory see page 97 Accessing Cavity Information for Relibase Database Entries see page 99 Displaying and Comparing Cavities see page 101 Cavity Similarity Searching see page 107 Saving Cavities to File see page 118 Building In House Cavity Databases see page 118 Introduction and Background Theory Introduction to CavBase CavBase is a program that can detect unexpected similarities amongst protein cavities e g active sites that share little or no sequence homology The program is supplied with a database of cavities from PDB protein structures including but not confined to known small molecule binding sites Any cavity or
92. Y DROXYPHOSPHINYLOXY 3 PHENYLPROPANOATE SODIUM SALT CRYSTALLOGRAPHIC ANALYSIS OF TRANSITION STATE MIMICS T pdbtppm 1 4963 5 83 4 33 0 528 100 0 90 2 HYDROLASE ACID PROTEINASE BOUND TO PENICILLOPEPSIN PHOSPHORUS CONTAINING PEPTIDE ANALOGUES CRYSTALLOGRAPHIC ANALYSIS OF TRANSITION STATE MIMICS m pdbtppk 1 4876 2 81 9 31 0453 100 0 92 7 HYDROLASE ACID PROTEINASE BOUND TO PENICILLOPEPSIN PHOSPHOROUS CONTAINING PEPTIDE ANALOGUES I ARID DDATEINAGCE DENIMI ADEDEINN EO 2 4 929 CARD EV AATU e To view the complete table of all 400 results click Browse all hits at the bottom of the table e One of the other options at the bottom of the page is Download Results Table in CSV Format Click this option and save the complete score ordered table as tutorial csv Return to the Cavity Comparison Search Result browser window e You will note that each matched cavity in the Cavity column of the table is hyperlinked to another webpage Click the link for the first cavity in the table pdb1bxq 1 e A Cavity Information page appears with some features of comparison between the hit cavity pdb1bxq 1 and the reference query cavity pdbIbxo 1 Click on the View in Hermes hyperlink at the top of the results page to view the cavities in 3D e Hermes will open along with a second smaller window the Cavity Controls window which you should move to one side of your screen leaving a view of the 3D display e You may w
93. Y_LICENCEDATA e Note that the paths and files specified need to be accessible by the machine that the Relibase Relibase User Guide 185 186 system is installed on Relibase PDB Entry Code Ligand Diagram Generation Configuration retibase 3 1 0 Enter the following information to configure Relibase to use Tripos SYBYL UNITY version 7 or above to generate ligand diagrams Options e View Current Settings TA_3DB home tripos sybyl x sybylx e Test Diagram Generation TA_LICENSE_FILE home tripose Admin Tools10 9 tables licence_file Submit TA_3DB the SYBYL installation directory e Tripos SYBYL e g homestripos sybyl 7 0 e ChemAxon MarvinBeans e OpenEye Toolkit TA_LICENCE_FILE the location of the SYBYL licence file or the port hostname of the licence server e Daylight Toolkit e g homestripos AdminTools9 2 tables license_file or 27000 myserver Configure With Manfred Hendlich 1994 1999 and Cambridge Crystallographic Data Centre 1999 2011 WS Cee Relibase User Guide APPENDIX E The Master Relibase Command If the Relibase environment is set see the installation guide for further information the command relibase is aliased to lt RELIBASE ROOT gt bin relibase master com The following actions and options are allowed Relibase Server Options relib
94. _ID 1 MOLECULE BETA TRYPTASE CHAIN A B C D EC 3 4 21 59 OTHER_DETAILS IN COMPLEX WITH AMIDINO PHENYL PYRUVIC ACID APPA A SYNTHETIC INHIBITOR e The bottom left frame displays the total number of hits found for the search To get a better impression of the type of hits that were found click on Browse Hit Headers This will list the headers of the entries that were hit e The places where the query string was found are highlighted in red Each protein entry code is linked to the corresponding Relibase entry page see Relibase Protein Entry Pages Section 4 1 page 27 Relibase User Guide 21 3 3 22 Relibaset Smiles Search Stored Results Keyword Search Results reivase 2 1 0 pdb1a01 pdblaoc pdb1a5h HEADER pdb1a5i pal pdbtaoc COMPND pdb1 ast COMPND pdbtatl SOURCE pdbtaugs SOURCE dbiaut SOURCE pabiovs pdbtazw pdbibda pdbibke pdblast pdbtbqb HEADER pdbibqq ey pabibay pdbibth pdbtbui pdbtbuv pablatl pdbic9t HEADER pdbicew pone pdbicgh comm pdbtcly COMPND pdbicsb COMPND pdbicse cmm pdbiczs SOURCE padbtezt pdbiczv sounce pdbtdtd AUTHOR 158 hits for query bode pabtaus Browse Hit Headers Saat COAGULATION FACTOR 28 Nov 96 1a0 c JAPANESE HORSESHOE CRAB COAGULOGEN MOL_ID 1 2 MOLECULE COAGULOGEN 3 CHAIN A B MOL_ID 1 2 ORGANISM SCIENTIFIC TACHYPLEUS TRIDENTATUS 3 ORGANISM_COMMON JAPANESE HORSESHOE CRAB A BERGNER V OGANESSYAN T MUTA S I
95. a Search Section 2 page 19 are displayed as three frames The top left frame contains the 2D chemical diagrams of the ligands that matched the query The right hand frame contains the Relibase ligand page see Relibase Ligand Pages Section 4 2 Relibase User Guide page 28 for the ligand currently selected from the list by default the right hand frame contains the ligand page of the first hit Ligands can be selected and inspected by clicking on the diagrams in the top left frame The following example shows the results of a ligand name search for amidin Relibase LOTA view Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Keyword Search Results relibase 3 0 0 server rc2 Hits 1 40 of 367 Similar Ligands Search Similar Ligands in CSD Similar Binding Sites Search Save Mol2 File Save SDFile Save Complex PDB File Save Complex Mol2 File Bookmark Ligand 120_246 PDB entry 1ghv Chemical name 2 2 0X0 1 2 DIHYDRO PYRIDIN 3 YL 1H BENZOIMIDAZOLE 5 CARBOXAMIDINE y Ligands v Chains 120 in pdb1ghy 120 in pdb1 ghz 120 in padbt gi y Solvent v Schematic Show Embedded Visualiser M width 500 Height pa Apply Hermes Controller Show in Hermes Automatic Visualiser U
96. ac uk products csd_system webcsd Only the 1000 most similar results to the query ligand are returned WebCSD Entry Identinier View ome ubstructure Searc Imi arity earc ex lumeric Searc educe e earc rowse ews eip H Sub S h Similarity S h TextiN ic S h Reduced Cell S h Bi CSD N Hel File Filter Help Licensed to CCDC at Cambridge Entry ACGLUAIO Hide Visualiser __Entry Similari gt GLUA10 0 89 Enel Details Viewer Export Options Help ACGLUA11 0 897 AGALAMO1 0 897 AGALAM10 0 897 NACMAN 0 897 ZZZAINEW 0 842 LILVUX 0 842 AMURAC 0 751 WEHSOR_ 0 732 BUBTIB 0 719 CAKFAV 0 719 0 708 oz 0 690 0 684 0 680 JAVUTUG 0 655 NAFHOR 0 652 NAFHUX 0 652 SUNFEM 0 649 LANEUME 0 638 YEQHEH 0 635 D SNIY 0 633 HN COMe Wiretrame gt NoLabels Ce His N Os lnn Lael Hydrogens Disorder Space Group P 2 1000 Hits Launch External Viewer a 11 250 30 b 4 820 10 9 720 20 o a 90 B 113 70 10 y 90 R Factor 10 4 Search complete Temperature K Room Temp 283 303 e The similarity index given in the left hand column adjacent to the 6 or 8 character CSD identifier 80 refcode is a Tanimoto coefficient Tanimoto coefficients are calculated from a comparison of topological fingerprints of each ligand against that of the reference ligand A fingerprint is calculated for each li
97. age Query Sketcher Results Results Results Click on a hit to view or browse all hits with the Browse button Ligand GGHECDIARM 1 X in entry pdbikat Model ID3 Parameter D2 3 70801 Ligand GGHECDIARM 1 X in entry pdbikat Model ID19 Parameter D2 3 77103 Ligand GGHECDIARM 1 X in entry pdbikat Model ID23 Parameter D2 3 83196 Ligand GGHECDIARM 1 in entry pdbikat Model ID9 Parameter D2 3 46962 Ligand GGNECDIARM 1 in entry pdbikat Model ID19 Parameter D2 3 85358 Ligand GGNECDIARM 1 in entry pdbikat Model ID23 Parameter D2 3 9861 Ligand GWLYEIS 1 B in entry pdbinlt Model ID1 Parameter D2 3 8545 Ligand KKETWY 420 B in entry pdbitp5 Model ID1 Parameter D2 2 66838 l Browse hits Close results pane Relibase User Guide e On completion of the search the protein entry browser see Using the Protein Entry Browser Section 3 2 page 20 or ligand browser see Using the Ligand Browser Section 3 3 page 22 will open automatically From here individual hits can be selected for viewing 6 8 1 Options available on Search Filters e There are several options are available in the Start search dialogue box These can be found on either on the Filters or the Hitlist Controls tabbed pages It is the Filters tabbed page that is displayed by default F Start Search Filters Hitlist Controls Hit Limits Structure filters Contact filte
98. age 131 can be made Relibase User Guide 131 5 3 variable by clicking on the bond type button at the bottom of the Draw window Select Variable from the pull down menu select the required bond types in the resulting pop up window then hit OK closes window or Add leaves window open The example below shows the setting required to create a variable bond type of double or aromatic Variable Bond Ty n a Bd Variable Bond Type _ Single Add v Double _ Triple Cancel v Aromatic Current definition 2 5 Java Applet Window Changing the Types of Existing Bonds This can be done in several ways including In any mode click on the bond with the right hand mouse button and select Set bond type from 5 4 132 the resulting pull down menu Then select the required bond type In Draw mode change the current bond type see Changing the Current Bond Type Section 5 1 page 131 and then click on the atom with the left hand mouse button In any mode click on Bond in the top level menu select Type from the resulting pull down menu and select the required bond type The Bond Property pop up appears click on the bond or bonds to be changed with the left hand mouse button and hit Done Defining Cyclic or Acyclic Bonds It is possible to specify that a particular bond must be cyclic i e part of a ring or conversely that it must be acyclic i e not part of a ring In Draw or Select mode click on
99. alue measures how much of the sphere volume is vacant i e unoccupied by protein atoms before the ligand is docked The latter measures how much sphere volume remains vacant i e unoccupied by either protein or ligand atoms after docking Volumes may be output in A or as percentages If percentage quantities have been requested the undocked value will always be 100 since by definition 100 of the sphere volume not occupied by protein atoms must be vacant before the ligand is docked If absolute A values have been requested the undocked value may vary a little from one docking to another because GOLD may move protein polar H atoms during docking therefore altering the amount of sphere volume that is occupied by protein atoms 196 Relibase User Guide APPENDIX G Tutorials Tutorial 1 Introduction to the Relibase Graphical User Interface see page 3 Tutorial 2 Substructure Searching in Relibase SMILES and 2D 3D see page 21 Tutorial 3 An Introduction to the Cavity Information Module and Hermes see page 31 Tutorial 4 Using the Cavity Information Module in a More In depth Way see page 43 Tutorial 5 Introduction to the Secondary Structure Module see page 59 nah ON Relibase User Guide Relibase User Guide 1 Tutorial 1 Introduction to the Relibase Graphical User Interface 1 1 Introduction e In order to familiarise yourself with the Relibase graphical user interface GUI it is recommended that you work t
100. an identical protein structure that has not undergone this conformational change Favourable interactions with the ligand provoke a large conformational change for residues in a section of sequence that is generally conserved over kinases This conserved section contains a Asp Phe Gly chain known as the DFG loop and the Phe residue previously buried in what now becomes a strongly lipophilic pocket moves by 10A to a position where the side chain occupies the space required by the phosphate groups of ATP e In this example we use the ReliBase cavity information module to identify the secondary structure characteristics of this rearranged DFG out region using the SHAFT protein classification We will also combine this with a 3D search to simply and quickly search the Relibase database for other examples of this DF G out conformation This is the sort of job one might wish to do when attempting to set up a pharmacophore for new inhibitors of this allosteric Relibase User Guide 61 5 5 62 site Secondary structure classification of the DFG region Open Relibase In the PDB Entry Code window on the top right of the Relibase interface enter 1 fpu The Protein Information page for the complex will appear The structure is a dimeric structure At the foot of the page is a table entry labelled Secondary Structure Information Click on this button A rotatable 3D image of the dimer will be displayed in the embedded 3D visualiser with s
101. and editing 3D and nonbonded contact parameters see Geometric Parameters Section 11 page 139 Area for changing the current bond type see Changing the Current Bond Type Section 5 1 page 131 Modes in the Drawing Window The four Mode buttons on the top left hand side of the Sketcher window are mode buttons which affect what happens when the mouse is used in the drawing area 120 Draw click on this button when you want to draw a substructure Select click on this button when you want to perform editing tasks such as moving or resizing substructures or selecting atoms or bonds Lasso as select but the selection area becomes a user defined shape rather than a rectangular panel Delete click on this button when you want to delete atoms or bonds Setting Molecule Types When using the sketcher you must ensure that the molecule type is set correctly for each substructure that is drawn There are three searchable molecule types in Relibase Protein Ligand and Water nucleic acids are not searchable Note In Relibase all moieties which are neither protein nor nucleic acid in a structure are considered to be ligands Hence metal ions anions solvate molecules except water cofactors and inhibitors are all regarded as ligands The molecule type Ligand must therefore be used when searching for these moieties Protein substructures are displayed in blue ligand substructures in black and water atoms in pink Relibase Use
102. ands and solvent can be switched on or off by clicking on the grey arrow adjacent to the PDB code e g pdblqs4 A_1 in the screenshot above e The display of components of the PDB entry protein chains ligands and solvent can be controlled by clicking on the relevant word adjacent to the green tick or red cross In the case of pdblk6y B_1 above the protein chains are displayed but the solvent is not e Use the Show All and Hide All buttons to control the global display e Control whether or not Astex Viewer is displayed via the Show Embedded Visualiser tickbox Relibase User Guide 45 5 1 4 Secondary Structure Display 46 Secondary Structure Information for PDB Entry 1raf Ligands L Chains C Solvent C Metals i Helices y Turns ASN A126 ALA A127 _ GLY A128 ASP A129_ GLY A130 SER A131 The display can be rotated translated and scaled as previously described see Astex ViewerTM Section 5 1 page 42 A number of check boxes beneath the display can be used to control the view Ligands displays or hides ligands Solvent displays or hides solvent molecules Packing displays or hides any crystal packing present in the protein Chains displays or hides protein chains Metals displays or hides metal atoms e Further details on controlling the display of secondary structure elements such as helices etc are provided elsewhere see Secondary Structure Information Section 4 6 page 35
103. aphic information e Viewer use this tab to configure the 3D viewer size and background colour e Export use this tab to output the structure currently on display as either a CIF an SDFile or a Mol file e Options use this tab to configure the 2D diagram display options e Help use this tab to access help on how to use the 3D viewer e The 3D viewer provided is Astex Viewer see Astex ViewerTM Section 5 1 page 42 Use the Hide Visualiser button to control whether or not the 3D view is shown Further basic options for controlling the display are provided at the bottom of the viewer e Display style use the pulldown menu that reads Wireframe to pick from Wireframe Capped Sticks Ball and Stick and Spacefill display modes e Display of labels use the pulldown menu that reads No labels to show labels for Selected atoms All but C H All but C H N O All Metals or All Atoms e Hydrogens tickbox use this to control whether or not H atoms are displayed if present on the CSD structure e Disorder tickbox use this to control whether or not disordered atoms are displayed e Use the Launch External Viewer button to view the structure in another visualiser 8 Similar Protein Chain Searches e On all Relibase entry pages the protein chains are listed at the bottom of the protein and ligand information chart Relibase User Guide 8l aes pdb1a01 B pdb1a01 C pdb1a01 D Nucleic F r j No nucleic acids for this entry Water
104. are coloured blue residues that are similar are coloured red and residues that are completely different are coloured black Check a few of the hits to see if the protein was isolated from the same type of electric ray Hitlist Manager Hitlists allow storage of query results on the Relibase server There are two types of hitlists protein and ligand depending on the type of input query For example author searches result in protein type PDB entry code hitlists whereas searches for ligand names result in ligand type hitlists To find all structures in the PDB where Bode is one of the authors and then store the results in a hitlist Click on the Text Search button in the Relibase menubar Change the Search Type option from PDB Entry Code to Keyword Type Bode into the Search String box Ensure the Search Field pull down menu reads Author Name Enter a hitlist name e g Bode into the Save in Hitlist entry box Hit the Submit button at the bottom of the page to start the search The results are presented as a browsable list of Relibase entries Relibase User Guide 11 Relibaset Home Text Search Sequence Search Smiles Search Sketcher Hitlists Stored Results Help Keyword Search Results reiibase 2 1 0 pdb1a0l pdb1a5h pdb1a5i pdbtaoc pdbtast pdb atl pdb1aus pdbtaut pdbtavg pdbtazw pdbibda pdbibke pdbibgb pdbibqq pdbibay pdbibth pdb tbui pdbtbuy pdbic9t pdbicew pdbicgh pdbicly
105. artial subsection of the cavity Specify search options and run the search View a hit cavity together with the query cavity in the 3D viewer using various viewer options to assess how well the two match Experiment with some other viewer settings 4 1 3 The Example 4 2 1BXO is an aspartic endopeptidase that is complexed with a strongly binding cyclic transition state mimic inhibitor PP7 Other examples of aspartic proteases include HIV protease and Cathepsin D An even closer sub family group are the BACE proteins which are strongly implicated in the progressive formation of insoluble amyloid plaques and vascular deposits consisting of beta amyloid protein beta APP in the brain e In this tutorial we will use CavBase to search for other ligands that might bind to this protein e We will then export these ligands into a mol file after they and their corresponding cavities have been superimposed onto a common reference frame e We will also show how CavBase can be used to search for and highlight movement of residues within a set of homologous structures Setting Up a Hitlist Open Relibase Relibase User Guide 43 4 3 44 When running a cavity similarity search we could if we wished search the whole database but that would take a long time To speed up the tutorial we will therefore search on a small subset that we know will contain enough examples of the CavBase features we need to highlight To do this we fir
106. ase all start Starts all Relibase servers relibase all stop Stops all Relibase servers relibase httpd start Starts the Relibase Apache HTTPD server relibase httpd stop relibase httpd shutdown Stops the Relibase Apache HTTPD server relibase database start Starts the Relibase Derby database server relibase database stop relibase database shutdown Stops the Relibase Derby database server relibase database status Reports the current status of the Relibase Derby database server relibase server start force Relibase User Guide 187 Starts the Relibase Derby database server Note the Derby database must be running before this command can be used The optional force command will force the Reliabse server to start even if it cannot detecet a running database server relibase server stop relibase server shutdown Stops the Relibase Derby database server relibase server status Details the current status of the Relibase server relibase software update Attempts to obtain and install the latest software update from the CCDC5 ftp server relibase software update package p ath_to_fi le Updates Relibase with the software update package specified by the path given in the package argument Relibase Data Commands relibase data update Attempts to obtain and install the latest data update
107. ase behind Relibase is the Protein Data Bank PDB Attp www rcsb org pdb It covers all entries in the PDB which were determined experimentally by means of X ray diffraction or NMR spectroscopy but not theoretical structures However structures where a ligand substrate molecule was modelled into an experimental protein structure are included In Relibase all non protein moieties in a structure are considered to be ligands Hence metal ions anions solvate molecules except water cofactors and inhibitors are all regarded as ligands In the 3D visualiser DNA and RNA strands are displayed as ligands but they are ignored in ligand substructure searches Database Entries Each protein entry in the Relibase database corresponds to an entry in the PDB and contains the following information see Database Information content Section 5 page 3 3 Bibliographic textual and numerical information Crystal structure data for X ray structures Protein chain s Binding site s Chemical diagram of the ligand s Crystal packing of the protein ligand binding site Water structure information Cavity information Secondary structure Entry codes Relibase uses the same entry codes as the Protein Data Bank PDB i e one digit followed by three characters e g Labe All modifications to the entry codes e g superseded entries reflect those made to PDB Relibase User Guide 4 Database Statistics A list of summary statistics n
108. ases gregtest ssetest Submit e Various Options are available e If you wish to display the ligand 2D chemical diagrams in the resulting list of chains select the Show Ligands check box this is the default Minimum Sequence Identity and Maximum Sequence Identity boxes can be used to specify the required sequence identity as a percentage with respect to the reference chain default is 100 e Save in Hitlist allows you to save the results of a search in a hitlist type the required hitlist name into the Save in Hitlist box before you start the search You will not be allowed to overwrite an old hitlist unless you click on the Overwrite Existing Hitlist button Note that it is not possible to save the search results to a hitlist after the search has been run e Use Hitlist allows you to to speed the search by restricting the data covered to a previously identified list which can be selected from the pop up menu next to Use Hitlist The default is Select existing hitlist until a hitlist is selected the entire set of databases will be searched e Similarly Use Databases allows you to select which database or combination of databases is searched The default setting is to search All databases e Hit the Submit button to start the search Relibase User Guide 65 e The results are displayed as a list of chains The ordering of the list is set using the Smith Waterman score This takes account of the sequence identity the number of re
109. at in addition to the atomic coordinates and residual sequence has appended a certain amount of secondary structure associated with each protein entry This secondary structure has been automatically assigned by software using methodology closely related to the DSSP algorithm of Kabsch and Sander DSSP recognizes eight types of secondary structure depending on the pattern of hydrogen bonds The 319 helix alpha helix and pi helix are recognized by having a repetitive sequence of hydrogen bonds in which the donor residue is three four or five residues later in the backbone In addition DSSP recognizes two types of hydrogen bond pairs in beta sheet structures the parallel and antiparallel bridge Schematic view on the hydrogen bonding network along a right handed a helix a 339 helix b antiparallel 8 sheet c and parallel 8 sheet Whilst these are the principle features recognised by the DSSP algorithm the procedure does recognise two additional turns in terms of H bonds but most commonly leaves other turns blank when no other rule pertains The SHAFT classification scheme is a new scheme published soon SHAFT Secondary Relibase User Guide 59 5 3 Structure Helix Assignment From Turns O Koch to be submitted It is based on the recently published turn classification O Koch G Klebe Proteins 74 353 67 2009 and uses a rather more consistent set of rules with regard to the termini of helical structures The turn classifi
110. ation 13 Low B factor 13 780 average 34 921 Octahedral coordination RMS 0 140 Short bonds lt 09A e General information on the water structure is given in the above table along with the criteria for notification of dubious water molecules see Data Related Issues Section 5 6 3 page 8 Information about water clusters and rings are also given The cluster or ring size is displayed along with the individual water molecules which form the structure Relibase User Guide 3 Water Clusters in Entry pdb1a3e Size _ Show Hermes r Et 15 la 44 25 m J 24 13 fafa 68 38 aoe 57 91 E show 163 19 Gma m Show 6 23 C Show 32 aco a show 117 31 120 51 99 H H EE 6 T Show 39 34 148 157 155 158 ace aire Sa HHHH Show 110 49 fasaa uaaa TT 5 show 20 so 129 ani 6 m Show 90 54 95 13 pel 133 87 W Oo aafe 61 65 E aL Ch I Show 103 66 Ee 3 Show 176 109 190 e H EE mE 113 41 H T 166 183 e Clicking on any of the hyperlinkable water molecules in the structure will lead you through to water descriptor information see Water Molecule Descriptors Section 5 6 page 4 e To view certain water clusters or rings in the embedded visualiser activate the appropriate tickbox underneath the column headed with the Show button All clusters rings can be viewed or hidden by hitting the Show button e To view certain water clusters or r
111. avities found by the cavity detection algorithm not just those of known significance Relibase User Guide 97 1 3 Cavity Detection 1 4 98 Cavities on the protein surfaces are detected using a modified version of LIGSITE see References page 172 which identifies surface depressions based on a grid based geometrical algorithm Very large cavities gt 3000A are omitted from the database as they are usually of little interest they are often ill defined gaps between large protein domains Some shallow or completely enclosed cavities are missed by the detection algorithm Pseudocentres A simple scheme is used for deriving 3D descriptors that encode the surface properties of all the cavities see References page 172 All amino acid residues lining a cavity are analysed to ascertain whether they determine the chemical property of the nearby surface This is based on geometric considerations e g a C O acceptor group pointing at the surface will be assumed to confer the property acceptor to the patch of the surface where the oxygen atom is exposed In contrast chemical groups pointing away from the surface will be neglected A dummy atom or pseudocentre is placed on the surface to represent the chemical property that is expressed by the atom s exposed in that area e g an acceptor patch of the surface would have an acceptor pseudocentre placed upon it A cavity is thus described by a set of pseudocentres each of which is
112. awing Substructures Select Edit in the top level menu and Undo in the resulting pull down menu to undo the last action performed If necessary Edit Undo may be used several times in a row to undo a sequence of actions one by one Selecting Atoms Selection of atoms is useful for setting the molecule type of atoms water ligand or protein deleting atoms moving substructures around the drawing area and for superposition of hits Selected atoms are shown in pink Relibase User Guide 123 File Edit View Atom Bond Options Help Query Sketcher 3d Parameters Left click in the selection box drag to move the selection C H O N S PF Cl Any Other N Ligand w Single Atoms and bonds may be selected in several ways 124 In Select mode an individual atom can be selected or deselected by clicking on it with the left hand mouse button In Select mode a series of atoms or bonds can be selected by clicking on each in turn while keeping the Shift key pressed down In Select mode a group of atoms and bonds can be selected by clicking with the left hand mouse button on a blank point in the white area and moving the cursor while keeping the mouse button pressed down Everything enclosed in the resulting rectangular box gets selected when the mouse button is released Groups of atoms within a non rectangular shape can also be selected in Lasso mode In any mode everything can be se
113. b3app 1 in reli Display Controls Search Setup Show Pseudocentres Query Matched PCs Hit Matched PCs v Link matching PCs with lines Separate cavities 7 e As a side issue you will notice that inspection of H bond contacts highlighted in this way on the back wall indicate apparently two quite short H bond contacts between the phosphonate fragment of the ligand and the acid residues Asp33 and Asp213 where one would not intuitively expect to see such an interaction One might speculate about the unseen presence of a Mg ion or other similar small cation You could even treat it as an excuse for another Relibase 2D sketcher search and analysis Relibase User Guide 53 4 6 54 It may be interesting to repeat the same exercise with the other low scoring 100 homologous protein structure 2wea Since this PDB entry contains a ligand structure very similar to that in 1bxo the real question might be to explain why the lack of collapse It might also be useful to look at the superimposition of the cavity 3cms 2 on our query cavity in the same way This protein which is only 30 8 homologous with our query structure and only a 39 cavity match 17 PCs matched out of 36 looks quite remarkably similar in some aspects Why Where might one suspect the mutational changes have been made Inspection of superimposed ligands When the initial CavBase search had been completed you may have noted that at the foot of the Cavity Compariso
114. base does not contain the target then relax the H atom specification to see if simple derivatives are present In an initial search do not over specify the substructure e g in terms of allowed substitution It Relibase User Guide 6 3 is better to get too many hits and then impose tighter chemical constraints Let the database tell you what it contains If you are unsure of the bond type used in the Relibase database for a particular substructure use the bond type any and look at the resulting hits in order to formulate a more precise query 3D Substructure Searching A 3D substructure search is one in which geometric constraints are added to a 2D substructure search so that only certain geometries are represented in the hits retrieved The geometric restraints may be either Distance Angle or Torsion Angle restraints Geometric parameters may be defined using just atoms or atoms and objects see Geometric Parameters Section 11 page 139 6 3 1 3D Ligand Substructure Searching Constraining geometry There are times when you wish to search for ligands which only conform to certain geometries For instance you may wish the two ends of the ligand to lie within a certain distance or you may wish to only find ligands which exhibit a certain type of intramolecular hydrogen bond 6 3 2 3D Ligand Substructure Searching Monitoring geometry 6 4 Another use for 3D ligand substructure searching is to monitor certain geometrical param
115. bile Ca helix and or an H bond acceptor contact with the same DFG Asp might produce a more extensive set of matches Try this search out if you wish o helical Glu a helical lipophilics ASP 1046 ASP1046 e If you are persuaded that filling the hydrophobic pocket newly created by the conformational change of the activation loop is essential for promoting this movement look at the structure of 2p21 e We have a suggestion that the DFG out conformation is frequently associated with a type 0 4 VII3 open turn Two other open turn types were also associated with the Phe and Gly residues of DFG in 1fpu 0 4 IX and 0 4 XID It might be worth redoing the constrained Relibase User Guide 69 substructure search again but this time make sure the Phe A382 is constrained to be present in all three turn types Do you get the same hits What secondary structure features appear in ABL kinase inhibitor protein structure which have DFG in not out You can investigate this by carrying out a Similar Binding Sites search starting from the lfpu Ligand Information page Looking for high homology inhibited structures will allow you to identify from the overlay of these structures other ABL kinase models with DFG in If you would rather not do this search have look at the secondary structures around the DFG region of protein ligand complexes 3dk3 3dk6 2hzi and f4j and apo structure 2g2i You should be able to identify for the inhibited stru
116. bstructures The following search shows how to find ligands comprising two or more different substructures A typical example could be the following task Thrombin has very distinctive specificity pockets The S1 pocket is well suited to accommodate basic moieties such as amidino groups whereas the S3 S4 aryl binding site preferably binds to large aromatic residues such as naphthyl groups Try to find all ligands containing both an amidino and a naphthyl group using both SMILES strings and the 2D 3D sketcher e Click on the Smiles Search button in the Relibase menubar Type the required amidino Smiles string into the Enter SMILES Code box Cc C ND ND and save the results of this search in hitlist amidino Relibaset Pes View Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Heip SMILES SMARTS Substructure Search relibase 3 1 0 Enter SMILES SMARTS Code Cc CEINDDIND e g NC Njctecceet Minimum MolWeight Maximum MolWeight Highest Resolution A j 0 Lowest Resolution A Structure Method Filters X ray V NMR M Exact Match SMILES I Similarity Search SMILES 7 Minimum Similarity p 3 Use Hitlist Select existing hitlist gt Save in Hitlist famidino Overwrite Existing Hitlist 7 All databases a davetest davetesttwo Use Databases Manfred Hendiich 1994 1999 and Cambridge Crystallographic Data Centre 1999 2011 vaci vac eg Relibas
117. by default have a resolution set of 1 0 and cannot be filtered in this way Structure Method Filters can be used to restrict the search to either X Ray or NMR derived structures The default is that no restriction is made Toggle off the criterion that is not required Use Hitlist allows you to to use a previously identified list which can be selected from the pop up menu next to Use Hitlist The default is Select existing hitlist until a hitlist is selected the entire set of database s will be searched Save in Hitlist allows you to save the results of a search in a hitlist type the required hitlist name into the Save in Hitlist box before you start the search You will not be allowed to overwrite an old hitlist unless you click on the Overwrite Existing Hitlist button Use Databases allows you to select which database or combination of databases is searched The default setting is to search All databases Relibase User Guide 4 Protein Sequence Searches 4 1 Performing a Sequence Search e Click on the Sequence Search button in the Relibase menubar e Type the required one letter code amino acid sequence into the Sequence Search box Enter Sequence 1 letter code Sequence must contain only standard amino acid symbols e g IVSWGEG IVSWGEG Show Ligands Vv Minimum Sequence Identity 100 0 Maximum Sequence Identity 100 0 Save in Hitlist C Overwrite Existing Hitlist O Use Hitlist Select existing hitlist Use Datab
118. cation required all the turns found in a non redundant dataset of 1903 protein chains to be clearly defined in terms of specific characteristics automatically clustered and then systematically classified One result of such an extensive classification is that not only are the commonly recognised helical and sheet structures identified but that the bulk of the remaining secondary structure much of which was hitherto unclassified is also classified in an objective systematic manner in terms of a relatively small number of turn types This includes a large number of interesting protein regions that previously could not be treated in this way eg catalytic serine protease triad DFG regions of tyrosine kinases etc Turn Classification recap As already mentioned in the previous section the recently published turn classification O Koch G Klebe Proteins 74 353 67 2009 is based on a non redundant dataset of 1903 protein chains The definition of the turn family is firstly based on a hydrogen bond between CO and NH n and then where there isn t an internal H bond on the Ca Ca distance subject to a distance constraint of less than 10 During the analysis following on from these definitions three different subcategories for turns based on the hydrogen bonding pattern between the first and the last residue have been introduced see the figure below a A reverse conformation with a hydrogen bond between NH and CO n b A standard or normal
119. cavities in XML format from the database lt db gt relibase dump_waters database lt db gt Exports all water information in XML format from the database lt db gt relibase dump_pdb_xml database lt db gt Exports all PDB data in XML format from the database lt db gt relibase hitlist_xml _ hitlist_uploa file d Imports the XML based hitlist file Pest acs asi 2 Fae 190 Relibase User Guide Relibase User Administrator Commands relibase licence_ info relibase check_licence Details information about your currently Relibase licence relibase workspace_edi tor Launches the Relibase workspace editor to allow administration of user workspaces This command will launch a java applet that will allow you to delete client workspaces relibase username password dpg_user_regi ster Enables an existing client workspace username to access the Data Processing GUI with the specified password relibase username dpg_user_chec k Displays the current details for the workspace username relibase username dpg_user_dele te Removes the Data Processing GUI rights for the specified username Note that the workspace username will still exist use the workspace editor to remove it relibase dpg_user_prin t Displays information for all users Relibase
120. cavity it is useful to display the ligand in space filling mode together with all or part of the cavity surface e By default carbon atoms in the query are shown in grey those in the hit in green Ligands are shown in stick mode protein chains in wireframe Different but related colour schemes are used for query and hit pseudocentres e g blue for query donors and cyan for hit donors red for query acceptors and pink for hit acceptors Aliphatic aromatic and pi pseudocentres are assigned the same colour by default since they are chemically similar Query metal pseudocentres are coloured orange while hit metal pseudocentres are coloured yellow 4 Cavity Similarity Searching 4 1 Overview of Cavity Similarity Searching Similarity searching allows you to find cavities or parts of cavities in the database s that match a query cavity or sub pocket The query cavity itself must be taken from either the CavBase database that is supplied as part of this release cavities from the PDB or from an in house cavity database that you have created The steps in similarity searching are e Load the query cavity into Hermes see Loading the Query Cavity Section 4 2 page 108 e Select which of the pseudocentres you want to search for see Selecting the Pseudocentres to be Searched For Section 4 3 page 108 e Select other search options e g how many hits to keep see Setting Search Options and Starting the Search Section 4 4 page 110 e Run the s
121. centres within a given distance of a pseudocentre click on the pseudocentre with the right hand mouse button and pick Select to range in the pull down menu Then select an appropriate distance e To select all pseudocentres within range of an atom that has not a pseudocentre superimposed or a bond right click on the atom or bond and then use the Select Pseudocentres within range of this ligand option You will be asked to enter a radius in the dialog window that appears e The box at the base of the Search Setup pane allows you to select pseudocentres of particular types The pull down options above the white box control how the pseudocentres are listed e g the settings below will cause pseudocentres to be listed firstly by whether they arise from backbone or side chain secondly by the pseudocente type and thirdly by the type of residue that they belong to Relibase User Guide Cavity Controls W Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1f0r 2 in reli Hit CavBase Hit pdb101m 1 in reli Display Controls Search Setup Use any of the methods below to select pseudocentres PCs in the Query cavity to be searched for Jaitclicking on PCs nghbalick on objects to pick nearby PCs clicking on the tick boxes below Use the pulldown options below to sortthe pseudocentres by diffe
122. cept the currently selected element types for drawing or OK to accept the currently selected element types and close the periodic table The resulting variable element type is called VZ if it is the first variable type created V2 if it is the second etc 4 3 Changing the Element Types of Existing Atoms This can be done in several ways including e In Any mode click on the atom with the right hand mouse button and select Set element type from the resulting pull down menu then select the required element type Other allows selection of some pre defined variable element types or selection from the periodic table see Setting Variable Element Types Section 4 2 page 128 In Draw mode change the current element type see Changing the Current Element Type Section 4 1 page 127 and then click on the atom with the left hand mouse button e In Any mode click on Atoms in the top level menu select Element from the resulting pull down menu and select the required element Other allows selection of some pre defined variable element types or selection from the periodic table see Changing the Current Element Type Section 4 1 page 127 The Atom Property pop up appears click on the atom or atoms to be changed with the left hand mouse button and hit Done e Select atoms and either click on Atoms in the top level menu select Element from the resulting pulldown menu and select the required element or right click on the canvas and select
123. ch If you look for this new list in the frame containing all stored hitlists you will find a Protein type tutorial hitlist containing around 200 entries Setting Up the Search Query At the top right of each Relibase window you ll notice a PDB Entry Code window PDB Entry Code In the PDB Entry Code window type 1BXO and press View Go to the very bottom of the protein information page for 1BXO and click on the Cavity Information button This will take you to the Cavity Information page for 1BXO where you will see that just one cavity has been identified for this protein structure and entered into the CavBase database Click on the link marked pdb bxo 1 Some additional information about 1BXO will appear Click on the Load in Hermes hyperlink to view the cavity in 3D Relibase User Guide e You ll notice immediately that the amino acids of the receptor are a little unusually for a PDB entry already protonated The view can be simplified by hiding the H atoms using the Hide H button at the top of the 3D display e If you rotate the cavity in the browser you will also notice that there is a long thin extension of the cavity down towards residue THR19 e By toggling the Active Surfaces off and on using the Graphics Object Explorer it can be seen that in this query structure this sub region of the cavity is empty e A first impression is that this might be the sort of region that could accommodate a lysine or an argenine residue Clo
124. ch Type Entry Code Search String torpedo californica Match whole words only I m Filter Search Options Minimum MolWeight Maximum MolWeight Highest Resolution A 1 0 Lowest Resolution A Structure Method Filters x ray M NMR M Use Hitlist esterase z Save in Hitlist Overwrite Existing Hitlist I All databases Use Databases Submit 6 4 Viewing and Editing Hitlists Click on the Hitlists button on the Relibase menubar This loads three frames into the browser below the menubar The top left frame lists the hitlists you have stored according to Set Name Owner Type ligand protein or cavity Size number of entries in hitlist and Access state Last modification date and time are also provided Set Name Owner berglig toby_iw_deb4 Ligand 6 Public Not owner tmp_1u4d adf_rb3 Protein 3 Public Not owner tmp_cav_1ud4 adf_rb3 Cavity 14 Public Not owner esterase henderson Protein 683 Private Change Delete hydrolase henderson Protein 10591 Private Change Delete The Access state indicates whether a hitlist has a Private or Public function Public hitlists can be viewed but not edited by other users Private hitlists can be neither viewed nor edited by Type Size Access Remove others Only list Owners can remove delete lists To view the contents of a hitlist as an ASCI or XML file click on the appropriate link under Content The hitlist will be displayed in the format spec
125. ch entry in the sixth column links to an expansion of the information available in the table The ligand pages of both reference and superimposed ligands can be accessed from here e Clicking on the header to column six links to a concatenation of the ligand overlap data for each chain All relevant chains are represented Protein chain PDB 1bbr E Overlap of ligands Superimposed Ligand yonma of Reference Reference Ligand vama of Superimposed 0 0 ACE DFLAEGGGY_308 F pdb1bbr 1 60 618 DX9 1 pdbifax_1 33 17 Protein chain PDB 1bhx F Overlap of ligands Superimposed Ligand Volume of Reference Reference Ligand Volume of Superimposed R56 1 L pdbibhx 1 59 113 DX9 1 pdbtfax 1 73 424 94 Relibase User Guide 9 4 7 The Analysis Table Conserved Waters e The seventh column of the table gives the number of conserved waters that have been identified within the region of the protein under consideration A conserved water is defined as one that is within 1 2A of a water in the reference binding site after superposition The column can be hidden from view in subsequent recalculations by clicking off the relevant checkbox in the Ligand binding site area at the base of the table e Each numeric entry in the seventh column links to a table that gives the residue numbers of the waters that are considered conserved in the superimposed structures The corresponding waters in the reference structure are also identified e The header
126. characterised by a property currently donor acceptor donor acceptor aromatic aliphatic pi and metal and 3D coordinates The rules for encoding pseudocentre assignment are given in the paper by Schmitt et al see References page 172 Additional pseudocentres have been assigned for the Relibaset implementation of CavBase e Pi type pseudocentres are used to represent the hydrophobic character above below amide guanidinium and carboxylate planes backbone Asn Asp Arg Gln Glu e Trp indole rings are represented by two aromatic type pseudocentres rather than one as given in the paper e Aliphatic pseudocentres can be assigned to the side chains of all amino acids The atoms taken into account for placing these pseudocentres do not include atoms of functional groups such as carboxylate For example for Glu the CB and CG carbon atoms are considered but not the carboxylate CD atom e Discrete metal atoms are treated as pseudocentres Note that metals of cofactors and metal containing ligands are not treated as part of the binding site and are therefore ignored by the pseudocentre generation algorithm Also note that there will be no Metal pseudocentre shown in the Visualisation Controls part of Hermes interface if there is no metal in the structure Relibase User Guide e All the information described above is precalculated and stored in a Relibase type database 1 5 Similarity Searching and Scoring e Similarity search
127. ching for Similar Ligands in the PDB Section 7 1 page 78 or on ligands stored in the Cambridge Structural Database CSD see Searching for Similar Ligands in the CSD Section 7 2 page 80 Note that the latter is available for CSD System subscribers only please contact admin ccdc cam ac uk for further information 7 1 Searching for Similar Ligands in the PDB e On any ligand page click on the Similar Ligands button on the menu bar above the 2D ligand diagram e All ligands in the Relibase database are compared to the reference ligand The results are loaded into the browser as a list of ligands and are ranked in decreasing order of similarity to the reference ligand e Only the 1000 most similar results to the query ligand are returned 78 Relibase User Guide Result of a 2D Similarity Search for THA_1 _pdb1mx1 Minimum Similarity 0 7 Submit Export XML Hitlist Save in Hitlist Save Search Results Hits 1 4 of 4 Similarity Index Ligand 1 000 0 702 0 702 e The similarity index given in the first column is a Tanimoto coefficient Tanimoto coefficients are calculated from a comparison of topological fingerprints of each ligand against that of the reference ligand A fingerprint is calculated for each ligand in Relibase by traversing each path of up to 10 atoms within the atomic graph At each atom in the path a standard hashing algorithm is then used to set 2 bits in a fingerprint of 2000 bits in length The fir
128. ciated with any pseudocentre will be displayed in mauve 3 3 5 Controlling Colour Schemes By default surfaces are coloured by pseudocentre i e each point on the surface is assigned the same colour as its nearest pseudocentre e Itis possible to change the colour of a pseudocentre type by right clicking the relevant type in the Graphics Object Explorer and selecting an appropriate colour from the pull down menu 106 Relibase User Guide 3 4 Tips Useful Cavity Viewer Settings Two overlaid cavities present a complex visual image When assessing how well a hit cavity matches with the query the following strategies may help e Start by switching the surfaces off and displaying the matched pseudocentres only to do this toggle on Active Surface and then toggle it off again Do for both cavities This should give you an immediate impression of how many of the query pseudocentres were matched and how closely e Switch the surfaces back on but undisplay all but one type of pseudocentre e g Donor This will hide all parts of the surfaces except those corresponding to the pseudocentre type that you have left switched on which makes it much easier to see how well the query and hit match Obviously this will need to be repeated for the other pseudocentre types Since the aromatic aliphatic and pi types are chemically very similar it is reasonable to inspect these parts of the surfaces together e To see how well a ligand fits into a
129. cise is to find other ligands that might bind to QacR so in this case we only want to find cavities that are occupied by ligands and ligands of a reasonable size Delete the 0 from the Minimum ligand size N atoms box and type in 2 instead This means that we will not find any cavities that do not contain a ligand of at least 12 atoms By default the search will keep the 100 most similar cavities that it finds irrespective of their score Leave these settings as they are Relibase User Guide 35 e We have a choice of scoring function There has been little work so far to investigate which is best so the choice between them is somewhat arbitrary In this example we will use scoring function 1 e The dialogue should now look as follows Relibaset Cavity Search Setup retitase 2 1 0 Similarity Search for Cavity pdb1jus 5 using 49 Pseudocentres Parameter Select an existing hitlist ix foutorial_ajus Search name Maximum permitted homology between proteins Minimum ligand size N atoms a CA Nanimum score to save scoring function 1 scoring function 2 scoring function 3 Number of solutions to save top N Select a Scoring Function Start Search Manfred Hendlich 7994 1999 and Cambridge Crystatiographic Data Centre 7999 2006 e Start the search by hitting the Start Search button e After a few seconds you will be shown a page that monitors the progress of the search
130. complete protein chains are aligned and the identity is calculated using only those residues in the cavity binding site Header header information taken from the PDB file Title the title record for the PDB file Relibase User Guide e A user specified list of cavities can be selected for display or for download using the tick boxes in the first column of the table Individual cavities can be selected by activating the corresponding tick box cavities can be selected or deselected globally using the Select button at the top of the column After the desired cavities have been selected use the Superpose Selected Cavity Binding Sites button at the bottom of the table to expose the following options e Display Superposed Cavity Binding Sites in Hermes click on this link to load the selected cavities into Hermes Download Superposed Cavity Binding Site in Mol2 Format click on this link to download the selected cavities to a mol2 format file If you change your mind and update your cavity selections use the Superpose Selected Cavity Binding Sites button to refresh the contents of the files to be displayed in Hermes or to be downloaded e To see more information about a hit click on the relevant cavity identifier in column 1 This will load both the hit and the query cavity into the 3D cavity viewer see Displaying and Comparing Cavities Section 3 page 101 and will also display two summary tables e g Comparison of cavities pdb1xie
131. control the display of individual pseudocentres Click on the icon for the Pseudocentres Aromatic branch in the hit cavity This will open the branch show all such pseudocentres in the hit cavity and their display state Pseudocentres for Phe 330 Tyr 124 and Tyr 341 should all be shown as displayed Individual pseudocentres can be hidden or displayed via the tick boxes in the tree This ends the tutorial 42 Relibase User Guide 4 Tutorial 4 Using the Cavity Information Module in a More In depth Way 4 1 Overview 4 1 1 Objectives To find ligands that might bind to a particular protein binding site This binding site of known interest will be used as the query cavity in a CavBase search for cavities with similar surface properties in the binding site If a hit cavity is sufficiently similar to the query then any ligand occupying such a cavity might also bind to the query cavity Such ligands may be of interest in their own right or may be the source of new ideas about possible hybrids or derivatives In addition where a series is being pursued as a consequence of activity in a known active site a CavBase search might highlight alternative binding Such binding might possibly be competitive and thereby detrimental to the efficacy of the putative drug candidate or possibly extend the spectrum of utility 4 1 2 Steps Required Prepare a hitlist so that we only search a subset of the database Set up a search query to match a p
132. correcting the naming of other crystallisation reagents such as glycerol and citrate which might have been inconsistently named over time Relibase also has the capacity to store structure factors with pre calculated electron density map coefficients which can be displayed as electron density maps in the web based viewer Structure factors can be deposited to Relibase along with the corresponding PDB file at the beginning of the data processing Relibase User Guide Relibase data processing has a web based graphical user interface as well as a command line interface The web based graphical user interface makes it easy to upload several structures at a time The command line interface makes it easy to process a large backlog of structures and to set up automated workflows for getting structures into Relibase 3 Ligand templates Ligand templates are required to ensure correct atom and bond typing of ligands as the PDB file format does not explicitly contain bond type information However having to validate templates of all ligands in all in house structures can easily become cumbersome The ligand template matching workflow has therefore been designed to minimise the amount of manual intervention required During the data processing the ligands are extracted from the PDB file The three letter code of the ligand is then used to check if an identically named template already exists in the main Relibase database reli This step is mean
133. ct match searches which will match on the given string only i e a search on Zet will not retrieve etr Note the text string is not case specific Relibaset PDB Entry Code view e Hit the View button to the right of the PDB Entry Code box to start the search e The results are presented as a single entry frame and the protein is displayed in a Hermes window if automatic visualiser updates are enabled 2 2 Performing an Entry Code Search using In House databases e Click on the Text Search button in the Relibase menu bar e Change the Search Type to Entry Code Type the required text string into the Search String box Select the database s to be searched in the Use Databases box and submit as before Entry code searches are not exact match searches and will find all matches that contain the search string Select the databases to be searched in the Use Databases box Relibase User Guide 57 2 3 Hints for Entry Code Searching e The searches are not case sensitive When using an in house database filenames can consist of underscores alphanumeric characters hyphens and must start with a letter either a z or A Z The separation of various parts of the filename for representation in the GUI is in the order of underscores first then digits and lastly hyphens for example ccdclmystructAal would be represented as Jmystructl ccdc and ccdc_lets_ MOI would be represented as Jets MQI ccdc Note regular expressions can be used
134. ctures one particular open turn which is not 0 4 VII3 and one or other of two closed turns all of which are associated with the DFG loop and adjacent Leu A384 and another very short and distinctive secondary structure element normally associated with Ser A385 and Arg A386 This ends the tutorial 70 Relibase User Guide
135. de for amino acids The searches are not case sensitive There are ambiguities in some entry codes of HET groups MAL for example is used for malonate anions but also for maltose As with ligand name searches it is often safer to use a substructure search instead Options for Text Based Searches Various additional Options are available for all text based searches However not all options are available for all searches Minimum Year and Maximum Year boxes can be used to restrict searches based on publication year Leaving the boxes empty means that all years will be considered To return hits from only one year e g 1995 enter 1995 into both Minimum Year and Maximum Year boxes These options are available for all searches Minimum MolWeight and Maximum MolWeight boxes can be used to restrict the molecular weight and size of ligands that you wish to retrieve from your search Leaving these boxes empty means that all ligands are considered These options are only available if a Ligand Compound Name or a Ligand PDB Code search is carried out Highest Resolution and Lowest Resolution boxes can be used to filter on the experimental precision of X Ray derived structures that you wish to consider If you only wish to consider X Ray structures with a resolution of 2 0A or better then enter 2 0 into the Lowest Resolution box If only the X Ray Structure Method Filter is toggled on then a Highest Resolution can also be set All NMR structures
136. ds from your current selection Since you want to investigate crystal packing for these similar binding sites ensure that the Get Crystallographic Environment check box is selected Packing tick boxes will then be present in the Hermes Protein Explorer Click on the Submit button to superimpose the selected chains Use Hermes to have a detailed look at all components of the various superimposed complexes As the Get Crystallographic Environment check box was selected there is an additional column of Packing check boxes which allow you to switch the crystal packing of the protein ligand binding sites on and off If there are no check boxes this indicates crystal packing isn t available If you select Ixka you will see that packing residues Lysine side chains bind in the S4 specificity pocket which is filled up by parts of the ligand in all other superimposed structures in Relibase User Guide 17 18 this example This indicates competition between the packing residues and the ligand in terms of interactions with the S4 specificity pocket Docking calculations carried out using GOLD indicate that it is possible for the ligand in 1xka to bind in a more extended conformation filling up the S4 pocket Competition for the S4 pocket is shown below Investigating crystal packing in this way provides a very quick and easy method of assessing which crystal structures are likely to exhibit packing effects simply by scrolling though the 3D visualiser
137. ds to extend helices to include additional residues most frequently at the C terminus end Visual inspection suggests that these alterations generally make sense For example in PDB entry 1fvt glutamine A131 is regarded as the C terminus of a 39 helix SHAFT extends the 3 9 helix to include residues ASN A132 and LEU A133 e Images of the 2 assignments can be seen below In the image one can also see that the SHAFT assignment has extended an helix by one residue This is also visible in the image below Original PDB Assignment SHAFT Assignment e Gamma Helices e SHAFT can assign helix status to regions previously deemed as parts of sheets which is well known in the literature This assignment is essentially subjective in practice the gamma helical status of the sheet strand is additional to its assignment as part of a sheet rather than an alternative as the gamma helix may still make the designated interactions that one associates Relibase User Guide 4 with a sheet An example of this behaviour can be seen in 1fvt A strand in the centre of a twisted beta sheet is reassigned as a gamma helix Original PDB Assignment SHAFT Assignment 5 3D Visualisation of Structures The two visualisers provided with Relibase serve slightly different purposes Astex Viewer see 5 1 AstexViewerTM Section 5 1 page 42 is embedded in the Relibase interface to provide quick and easy visualisation of hit structures including the disp
138. e User Guide 21 22 Click Submit to run the search Repeat the search for naphthyl and type the required Smiles string for naphthyl into the Enter SMILES Code box clcc2cceccec2cc and save the results of this search in hitlist naphthyl Click on the Hitlists button on the Relibase menubar and combine the two hitlists you have generated AMIDINO and NAPHTHYL Generate a new hitlist using the AND operator by selecting AMIDINO from Ligand Set 1 and NAPHTHYL from Ligand Set 2 e g NAPAM Note down the numbers of hits in each of these hitlists including the new one you have just created combining the two hitlists Look at some of the hits to observe what type of hits are being retrieved Click on the Delete link next to MAPAM in the column labelled Remove to remove the combined hitlist Repeat the substructure searches using the 2D 3D sketcher Click on the Sketcher button on the Relibase menubar to take you into the Relibase sketcher Ensure that the molecule type is set to Ligand and draw the amidino group attached to a carbon atom as shown below Relibaset Pe View Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help 2D 3D Sketcher retibase 3 1 0 File Edit View Atom Bond Options Help Query Sketcher 3d Parameters alr l Click to place atom click and drag to draw bond Il Add 3d 3d ii oo X1 we i Choose lt View
139. e Residues Only Get Crystallographic Environment 7 Keep Reference Ligand Position M Superimpose p3 Selected Chains 2 seconds per selected chain Submit Filters Reset Selection Invert Selection First Chain Per Entry With Ligands Only Advanced Filters Minimum Chain Length aa Minimum Alignment Identity Select Chain overlap Ligands Z score 100 M pdbtacl A 537 aa 4407 7 0 o LA w 100 M pdbtut6 A 537 aa mA 4407 7 L Z No O O i 100 hS Z Es T ndhtamn iM 535 as S gt e The links from the percentage sequence identity in this table will show the alignment of two complete chains e Use the check boxes in the left hand column of the table to select or deselect the chains for 3D superposition If you switched on the Preselect Protein Chains check box prior to searching all chains will be selected automatically Various options are available for chain selection e Reset Selection returns you to the original chain selection i e that chosen for initial viewing of 86 Relibase User Guide the results e Invert Selection allows you to toggle back and forth between selected and deselected chains in the results list e First Chain Per Entry only the first chain is used for superposition if the protein contains more than one chain from your current selection e With Ligands Only selecting this allows you to exclude entries with no ligands from your current selection e Min
140. e Save Superposition Results or Save Sequence Results part of the window generally found at the bottom of the page as well as a description of the search then hit Save Save Superposition Results Name Description Save Overwrite Existing Superposition I For text searches hit the Save Search Results button on the bottom right of the page enter a name for the search results then hit Save Relibase User Guide 47 183 hits for query bode Browse Hit Headers Save Search Results e Stored searches i e for searches run in batch mode see Options available on Search Filters Section 6 8 1 page 75 can be accessed from the Stored Results window Cavity similarity searches are saved automatically and can also be viewed Relibase eT View Text Search SMILES Search Sketcher Hitlists Stored Results Help Stored Results Relibase 3 0 0 server rc2 Stored Search Results seg Searcy bode_authr a Status Finished Result Description bode Update Owner henderson Access Private Change Display Delete Stored Superpositions No stored Superpositions Stored Similar Sequence Results Select Similar Sequence Result simsearch toby_ff_xp v Status Finished Description smiles subculture search Owner toby_ff_xp Access Public Display Stored Similar Cavity Results Show Similar Cavity Results e Itis not possible to edit combine or manage stored search results 6 2 Creating Hitlists e Sea
141. e Search String box Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Text Search relibase 3 0 0 rcs Search Type Keyword bd Search String famidin Match whole words only I Use regular expressions Keyword Search Options Search Field Ligand Compound Name X Filter Search Options Minimum Year Oo Maximum Year Eoo o i Minimum MolWeigt Maximum MolWeight Poo Highest Resolution A 10 Lowest Resolution A Structure Method Filters X ray M NMR M Use Hitlist Select existing hitlist Save in Hitlist amidin Overwrite Existing Hitlist Submit e Various additional Options are available for all text based searches However not all options are available for all searches see Options for Text Based Searches Section 3 5 page 64 Relibase User Guide 6l Hit the Submit button to start the search The results are presented as a browsable list of ligands The search text will be highlighted in red within the Chemical name of each ligand The ligand and corresponding binding site are displayed in the Astex Viewer window The search results can be saved to a hitlist after the search has been run using the Save in Hitlist button in the bottom left hand frame of the results window 3 3 2 Hints for Ligand Name Searching 3 4 The searches are not case sensitive Ligand name searching can be useful as a quick way of finding examples
142. e Start search window go into the Hitlist Controls tab and type AMIDINO into the Restrict search to hitlist named entry box and then BIDENT into the Save search in hitlist named entry box this will save all hits corresponding to bidentate salt bridges Select the Start button to start the search Look at some of the hits in Hermes to ensure that you have retrieved the expected results click on the Show in Hermes button if Automatic Visualiser Updates is not activated the atoms included in the parameter definition are indicated in the visualiser using a van der Waals surface The results of the search matching groups can be superimposed onto a set of selected atoms Relibase will use all atoms selected prior to submission of the query for superposition Investigate how the amidino groups bind to the carboxylate group look at the orientation of binding and use carboxylate as a reference for superposition see which contact distance is the most common Return to the query ensure the sketcher is in Se ect mode and select 3 atoms of the protein carboxylate group by pressing Shift and left clicking on the atoms Alternatively depress the left mouse button and drag a rectangle to encompass the 3 atoms of the carboxylate group Selected atoms are shown in red in the drawing area Click Search In the Start search window select the Superimpose hits on selected atoms check box to generate an overlay of the hits Selection of this check box generates
143. e_structurel pdb This entry will be deleted from the database when the Delete button is selected 7 5Cancelling work in progress e If halfway through processing your structures you for some reason or other want to cancel the data processing this can be achieved by clicking on the Cancel hyperlink on the left hand side of the data processing page 8 Processing structures using the command line e Structures can be added to Relibase using the command line option below Further information is available see APPENDIX E The Master Relibase Command Section page 187 168 Relibase User Guide relibase data_process input filename_or_dir database specification conf configuration_file mt z mtz_ file template template_file 8 1Processing a single entry e Suppose that you had the files 2BYH pdb 2BYH_2D7 mo12 the ligand template file and 2byh_sigma mtz and you wanted to add the entry to a database named aaa then you could simply use the command relibase data_process input path to dir 2BYH pdb database aaa mtz path to dir 2byh_sigma mtz template path to dir 2BYH_2D7 mol2 e This would add your structure to the database e Note that the MTZ and template flags are optional However if you do not provide a ligand template the structure will not be entered into the database if that template needs user validation Instead you will get a message stating that There are ligand templates that require confi
144. earch Relibase User Guide 107 4 2 4 3 108 Loading the Query Cavity Load the query cavity into the viewer by clicking on an appropriate link in a browser page see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 Move to the Search Setup pane of the viewer using the tab at the top right of the Cavity Controls window Selecting the Pseudocentres to be Searched For When doing a cavity similarity search it is not necessary to include all of the query cavity pseudocentres in the search For example you can select just the pseudocentres in a sub pocket during the similarity search all the other query cavity pseudocentres will be completely ignored i e the software will just try to find a good match for those in the sub pocket This is useful for example if you just want to find a good match for the immediate environment of a ligand that only occupies part of the query cavity The selection of which query cavity pseudocentres are to be included in the search is done by using the Search Setup pane of the Cavity Controls window Unselected pseudocentres are displayed as translucent picked pseudocentres are displayed as solid The surface patch corresponding to each picked pseudocentre will be displayed Pick the pseudocentres you want included in the search in any of the following ways e Click on a pseudocentre with the left hand mouse button to toggle its selection state e To select all pseudo
145. earch Section 6 8 page 74 the resulting overlay of hits is displayed in the embedded visualiser window see Astex ViewerTM Section 5 1 page 42 Alternatively the 3D superposition can be read into Hermes using the Show in Hermes button 26 Relibase User Guide 4 Viewing Information for Individual Hit Structures 4 1 Relibase Protein Entry Pages Each Relibase protein entry page contains Relibase User Guide Embedded 3D visualisation via Astex Viewer see Astex ViewerTM Section 5 1 page 42 A Hermes control panel see Hermes Section 5 2 page 47 Protein and ligand information see Protein and Ligand Information Section 4 3 page 30 A link to information on water structure in the entry see Water Information Section 4 4 page 31 A link to information on cavities in the entry see Cavity Information Section 4 5 page 34 A link to information on the secondary structure in the entry see Secondary Structure Information Section 4 6 page 35 Customisable content and hyperlinks to external resources can also be added see the Relibase Installation Notes Appendix B http www ccdc cam ac uk support documentation relibase Relibase eer view Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Protein Information for PDB Entry 1bhxX retibase 3 0 0 server rc2 View PDB Header Save PDB File PDB Website Bookmark Protein Entry 1bhx
146. ect which database or combination of databases is searched The databases must first have been loaded when the Relibase server was started see Section 3 of the Inhouse Data Processing manual The default setting is to search AlI databases e Hit the Submit button to start the search The results are presented as a browsable list of Relibase entries The keyword searched for is highlighted in red e Browse thought the hits select the Browse Hits hyperlink From the resulting list search and locate the structure of acetylcholinesterase from Torpedo Californica an electric ray e g PDB entry le3q Relibase User Guide 9 Header SERINE HYDROLASE Title TORPEDO CALIFORNICA ACETYLCHOLINESTERASE COMPLEXED WITH BW284C51 Compound MOL_ID 1 MOLECULE ACETYLCHOLINESTERASE CHAIN AEC 3 1 1 7 Reference ACTA CRYSTALLOGR SECT D 58 2002 1765 Author s C E FELDER M HAREL SILMAN J L SUSSMAN Source MOL_ID 1 ORGANISM_SCIENTIFIC TORPEDO CALIFORNICA ORGANISM_COMMON PACIFIC ELECTRIC RAY ORGAN ELECTRIC ORGAN TISSUE ELECTROPLAQUE Method X RAY DIFFRACTION Crystal Cell a 114 000 b 114 000 c 158 200 alpha 90 000 beta 90 000 gamma 120 000 Spacegroup P3121 Resolution 2 850 RFactor Unavailable Deposition Date 20 6 2000 Ligands ps N 0 0 NAG in PDB Entry 1e3q Water Mediated Protein Ligand Contact Paths 1 1 4 3 Similar Chain Searching Acetylcholinestera
147. eight 350 Apply e In addition to the 3D display a 2D diagram is also provided The display can be rotated translated and scaled as previously described see Astex Viewer TM Section 5 1 page 42 A number of check boxes beneath the display can be used to control the view Ligands displays or hides ligands Solvent displays or hides solvent molecules Metals displays or hides metal atoms Chains displays or hides protein chains Packing displays or hides any crystal packing present in the protein Schematic displays or hides the protein cartoon display Note the schematic is assigned by Astex Viewer and not Relibase nor the PDB e Whether or not Astex Viewer is displayed is controlled via the Show Embedded Visualiser 44 Relibase User Guide tickbox The dimensions of the viewer size default is 500 by 350 may also be modified to fit user requirements by typing new dimensions into the Width and Height windows then hitting Apply 5 1 3 Appearance of Astex Viewer on a Binding Site Superposition Page pdb1qs4 A_1 a x ligand chains X solvent pdb1biu C_1 x chains X solvent X solvent pdb1bis B_1 x chains X solvent x solvent pdbtgs4 C_1 z Show All Hide All Show Embedded Visualiser M e The 3D display contains the superimposed binding sites while the pane to the right controls the view in the 3D display e An entire PDB entry including protein chains lig
148. ein 1 3 The Relibase Home Page Relibase is a web based application and all its functionality is accessible via a web browser such as Netscape or Internet Explorer Open Relibase from within your browser Relibase User Guide 3 The 3D visualisation software will already have been installed for you Two visualisers are Relibaset Home Text Search Sequence Search Smiles Search Sketcher Hitlists Stored Results Help Homepage relibases 2 2 0 databases 30 Vi t 3 re Cliem Workspace Administration In house Database Bulang Tool Cavity Similarity Results Comments bugs quenes to S 12 Union Road Camonage C52 1EZ United Kingdom OD ier 44 1223 236022 ww ftp Aww code cam se uk emal suppami cede cam ac uik Relibase A program for searching protemigand Cambridge Crystallographic Data Centre Current workspace robertson Databases currently loaded reli R Powered by Diagram generation in Relibase is powered by ChemAxon s Marvin Softw E chemixon Oastex D Mantred Henalich 1994 1999 and Cambridge Crystallographic Data Centre 1999 2007 Embedded visualisation in Reibase is powered by AstexViewer provided with Relibase and serve slightly different purposes e AstexViewer which is embedded in the Relibase interface to provide quick and easy visualisation of hit structures e Hermes to facilitate more detailed investigation of hit structures The workspace userna
149. elected the entire set of databases will be searched e Save in Hitlist allows you to save the results of a search in a hitlist type the required hitlist name into the Save in Hitlist box before you start the search Activate the Overwrite Existing Hitlist tick box if you wish to overwrite a hitlist already in existence Note it is not possible to save a hitlist of sequence similarity search results after the search has been run however search results can be stored see Storing Search Results Section 6 1 page 47 e If you wish to display the ligand 2D chemical diagrams in the resulting list of chains select the Show Ligands check box Note selecting this option may adversely affect the search speed e Hit the Submit button next to Search for Similar Chains to start the search The results are displayed as a table of chains ranked according to their sequence identity relative to the reference chain 98 6 141 aa pdb1011 A e The links from the sequence identity in this table will show the alignment of two complete chains e g Alignment of Entries pdb101I A and pdb1a01 A pdb101I A 283 Amino Acids pdb1a01 A 141 Amino Acids Identity 98 6 LPAEFTPAVHASLDKFLASVSTVLTSKYRGVLSPADKTNVKAAWGKVGAHAGE YGAE AWE VLSPADKTNVKAAWGKVGAHAGE YGAEALE RMFLSFPTTKT YFPHFDLSHGSAQVKGOGKKVADALTNAVAHVDDMP NALS ALSDLHAHK RMF LSFPTTKT YFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALS ALS DLHAHK LRVDP VNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVL
150. en bonds to the protein i e via a water molecule and measure a few H bonding distances Also highlight all the short nonbonded contacts in the binding site Protein H Bonds Short Contacts 1 THA_999_PDB1AJv v e Repeat the PDB entry code search for PDB entry 1qs4 Hyperlink through to the ligand information page and inspect the structure using Hermes as before This time you will notice that there are columns present for Metals and Packing in the Protein Explorer window in Hermes Relibase User Guide M AllEntries vw Ww WM Ww HM 100 1004 PV WV VM VW N e Packing is switched on by default look at the packing of the ligand in the binding site 1 4 2 Text Searching Text based searches are a convenient tool for retrieving sets of ligands or proteins However please keep in mind that due to inconsistencies in annotation sequence or substructure searches are usually a better means of getting comprehensive information out of the database e In order to find a number of acetylcholinesterase structures click on the Text Search button in the Relibase menubar e Select Keyword from the Search Type pull down menu then ensure the Search Field is HEADER TITLE COMPND and SOURCE Records e Type the required text string into the Search String box i e acetylcholinesterase e Various options are available for all text based searches 8 Relibase User Guide e Minimum MolWeight and Maximum MolWeight boxes can be used to restr
151. ent is given for each ligand Note It is not recommended to use the bond type any Symbol or the three atom wildcards A R and X in similarity searches as the matching of these symbols is not supported for that use 6 2D 3D Ligand Substructure Searches 6 1 70 2D Substructure Searching A 2D substructure search is most usually carried out to find ligands which include the 2D substructure that is of interest There are four molecule types in Relibase Protein Nucleic Acid Ligand and Water A 2D substructure search must be used to find proteins with unusual amino acids The definition of a Protein in Relibase encompasses normal ie the 20 most commonly occurring AAs amino acids only Obviously most protein substructure search that do not involve these unusual residues can be more easily carried out via a Sequence Search see Performing a Sequence Search Section 4 1 page 65 Nucleic acids cannot be searched via a substructure search A 2D ligand substructure search is often worth doing prior to a 3D substructure search in order to reduce the size of the search list that will be queried by the 3D search Hints for 2D Substructure Searching If you are unfamiliar with chemical substructure searching try a few simple searches e g for 6 membered carbocyclic rings 4 coordinate transition metals Finding exact structures requires complete definition of the target molecule including H atoms If the Relibase data
152. entries can be e Added to a different hitlist select the target hitlist from the popup menu next to Add to Set and hit the Submit button e Removed from the current or from another hitlist select the target hitlist from the popup menu next to Remove from Set and hit Submit e Make into a new hitlist enter the name of the new hitlist into the text window next to Make New Set and hit Submit Relibase User Guide 6 5 Combining and Translating Hitlists e Click on the hitlists button on the Relibase menubar This loads three frames into the browser below the menubar In the bottom left frame you can combine or convert different hitlists using logical operators in order to generate new hitlists Hitlist Operations for User henderson Ligand Set 1 Operation Ligand Set 2 New Set Select existing hitist AND Selectexstngnitist Submit Protein Set1 Operation Protein Set 2 New Set Select existing hitist AND Selectexistinghitist gt Submit Cavity Set 1 Operation Cavity Set 2 New Set Select existing niist AND Le Selectexstngnitist E gt Submit Database 1 Operation Set2 New Set All databases regtest MINUS Select existing hitlist gt Submit Read Hitlist Format XML X Browse Submit e To combine hitlists using simple logical operators e Select the two hitlists you wish to combine using the pull down menus below the appropriate hitlist type e g Ligand Set I and Ligand S
153. er Guide 19 3 Viewing and Navigating Search Results 3 1 Overview Relibase searches started from the Relibase menubar i e Text Search Sequence Search SMILES Search and Sketcher searches see Starting a Search Section 2 page 19 can result in a three different browsable lists of hits e A list of Relibase entries see Using the Protein Entry Browser Section 3 2 page 20 e A list of protein chains see Viewing Sequence Based Search Results Section 3 4 page 24 e A list of ligands with their binding sites see Relibase Ligand Pages Section 4 2 page 28 To view the results of other types of searches started from Relibase protein entry pages see Relibase Protein Entry Pages Section 4 1 page 27 or from Relibase ligand pages see Relibase Ligand Pages Section 4 2 page 28 the user is referred to the sections where these searches are described 3 2 20 Ligand similarity searching see Similar Ligand Searches Section 7 page 78 Similar chain searching see Similar Protein Chain Searches Section 8 page 81 Similar binding site searching and superposition see Similar Binding Site Searches and Superposition Section 9 page 84 Using the Protein Entry Browser Search results from text searches see Keyword Searches Section 3 page 58 and author searches see Author Name Searches Section 3 2 page 59 are displayed as three frames The top left frame contains the protein entry codes of the hits The right
154. erence pdbtacj A Residues 528 Protein Chain Parameters Minimum Sequence Identity so 7FCt S Maximum Sequence Identity hooo Search Parameters Lowest Resolution A DO E Options Preselect Protein Chains M Show Ligands M Use Hitlist Select existing hitlist Save in Hitlist Overwrite Existing Hitlist I All databases davetest davetesttwo etr_test reli gt Submit Use Databases e Using the radio buttons next to the chain identifiers select the chain you wish to use as the reference chain The reference chain is the chain that will be used for the sequence alignment and for the 3D superposition e If required change the sequence identity limits using the Maximum Sequence Identity and Minimum Sequence Identity text boxes e If required enter a resolution limit e g 2 0A into the Lowest Resolution text box e Further options are available e If you wish all chains included in the 3D superposition to be preselected in the list of results ensure that the Preselect Protein Chains check box is switched on this is the default otherwise switch off this check box and then make your selection from the list of results e If you want the ligand diagrams to be displayed in the resulting table of chains make sure the Show Ligands check box is selected e Use Hitlist allows you to to restrict the search to a previously saved hitlist Select the hitlist name from the Use Hitlist pulldown menu The default
155. ery esterase Export XML Hitlist Browse Hit Headers e In the case of similar binding site searches results can be saved after a search has been run by typing the hitlist name into the Save in Hitlist box at the bottom of the similar binding site search results page then hit Save Save Hitlist Save in Hitlist Save Overwrite Existing Hitlist I e The hitlist type protein ligand or cavity will be determined automatically according to the search being run 6 3 Using Hitlists in Subsequent Searches e The following searches can use hitlists which have been saved from a previous search Text searches see Keyword Searches Section 3 page 58 SMILES searches see Ligand SMILES or SMARTS Searches Section 5 page 66 2D 3D searches see 2D 3D Ligand Substructure Searches Section 6 page 70 Sequence searches see Protein Sequence Searches Section 4 page 65 Binding Site Superposition searches see Similar Binding Site Searches and Superposition Section 9 page 84 e To use a previously saved hitlist select the required hitlist from the drop down menu next to Use Hitlist Only entries in the selected hitlist will be considered in the new search e The example below shows a keyword search for entries in the protein hitlist ESTERASE 50 Relibase User Guide Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Stored Results Text Sea rch Relibase 3 0 0 server rc2 Sear
156. es Aliphatic O Pseudocentres Aromatic E Pseudocentres Donor a O Pseudocentres Donor Acceptor E3 Pseudocentres Pi H Relibase User Guide 3 3 2 Controlling which Molecules and Residues are Displayed e The Protein Explorer window usually found to the top left of the hermes display can be used to control the display of individual ligand molecules waters and proteins The display of chains within proteins and individual amino acids residues can also be controlled please refer to the Hermes documentation for further information 3 3 3 Controlling which Pseudocentres are Displayed e The tick boxes in the Graphics Object Explorer window bottom left of the Hermes window can be used to switch on or off the display of particular types of pseudocentres see Pseudocentres Section 1 4 page 98 you may need to click on the icon next to P Centres to list the separate types e Ifthe viewer is showing both the query cavity and a hit cavity from a similarity search the display of pseudocentres may be further restricted to convey information about which pseudocentres in the query matched which in the hit and which query pseudocentres were unmatched see Displaying Matching and Non matching Pseudocentres Section 3 2 3 page 103 3 3 4 Displaying Parts of Surfaces e First some background information by projecting each pseudocentre onto the cavity surface the surface can be partitioned into patches each patch ass
157. es as a reduction of the 100 homology noted for the complete protein chains to 90 e Because the initial search was limited to structures with a known strong functional similarity to the query receptor it is not surprising that the region of primary interest that part of the plot where there is low cavity homology but high PC matching is disappointingly vacant e Suffice to say that most of the hits found in this region of the plot above are BACE enzymes which themselves are part of a cluster of strongly conserved structures with a well known partial similarity to the penicillopepsins Relibase User Guide 57 4 8 Superimposed CavBase cavities obtained from 1bxo and 2web ASN117 e Although these these two cavities superimpose very closely RMS 0 56 Angstroms you will notice the four extra labelled amino acids in the 40 residue query 1bxo green at the bottom of the figure which are not matched in the corresponding 2web hit yellow and which account for the resultant cavity homology of just 90 58 Relibase User Guide 5 Tutorial 5 Introduction to the Secondary Structure Module 5 1 5 2 Objectives To use the protein secondary structure database SecBase its turn classification and its SHAFT helix assignment H bonding classification to probe possible associations with turn elements and special modes of kinase inhibition SHAFT classification recap Relibase has historically stored structural files in a pdb format th
158. es information on significant C alpha movements if any with respect to the reference chain The default threshold for what constitutes a significant movement is 0 5A This threshold can be changed by altering the relevant figure in the C alpha Movements box in the Protein flexibility area at the base of the table and then clicking Recalculate Table The column can also be hid from view by clicking off the appropriate check box in the Protein flexibility area prior to recalculation e Each numeric entry in the C Alpha Movements column links to an expanded list of residues involved in movement and the distance of movement in each case Relibase User Guide 9 Protein chain PDB 1e66 A Movement of C alpha atoms Residue Distance SER 4 1 688 GLU 5 0 668 VAL 22 0 658 LEU 23 0 883 SER 24 0 831 SER 25 0 618 SZAL 74 naa e The header of the third column links to a summary table of C alpha movements for all chains in the Analysis Table Movements of greater than 1 0A are highlighted in red C alpha Movements Reference chain PDB 1acj Reference ligand THA_999_pdb1acj_1 Note values above a threshold of 1A are highlighted in red Protein Chain PDB 1e66 A PDB 1ea5 A PDB 1gpk A PDB 1gqr A PDB 1h22 A PDB 1h23 A 1 513 PDB 1ode A 1 815 DNR taidA iM 1 A99 SER4 VAL22 LEU23 SER24 SER25 VAL71 PRO76 GLY77 LEU146 SER147 GLY160 GLU261 ILE347 HIS362 ALA363 1 688 1 498 1
159. et 2 The hitlists to be combined must be of the same type i e either both Ligand both Protein or both Cavity e Select the logical operator to be applied on the two hitlists AND means each entry in the new hitlist must occur in both hitlists OR means each entry in the new hitlist must occur in at least one of the two hitlists MINUS means each entry must occur in the first hitlist but must not occur in the second e Enter the name of the new hitlist in the appropriate New Set text box and hit the Submit button to create the new hitlist e Hitlists of entries can be converted from one type ligand protein or cavity to another Convert hitlists as follows e Select the hitlist you wish to convert using the popup menu below either Ligand Set 1 Protein Set 1 or Cavity Set 1 e Select gt Ligand gt PDB or gt Cavity where appropriate from the popup menu of logical operators note that the menu options available reflect the hitlist type selected e Enter the name of the new ligand protein or cavity hitlist in the New Set text box and hit the Submit button to create the new hitlist e Hitlists can also be copied or renamed e Select the hitlist you wish to copy or renamed using the pulldown menu below either Ligand Relibase User Guide 53 Set 1 Protein Set 1 or Cavity Set 1 e Select the Copy or Rename option from the corresponding Operation pulldown menu e Type in the new hitlist name for the copied or renamed list and S
160. eters This is useful for e g e Generating geometry histograms e Locating substructures with specific conformations torsion angles e Locating specific metal coordination geometries e g tetrahedral rather than square planar Geometry histograms can be accessed once a search is completed When a 3D substructure search is completed a histogram s link appears at the bottom left hand corner of the Substructure Search Result page Clicking on this link brings up histograms of frequency against geometry for the geometry parameters defined in the search see Viewing Distribution Histograms for Geometrical Parameters Section 3 5 1 page 25 Basic Guide to 3D Ligand Substructure Searching Draw the required substructure See CHAPTER 5 USING THE RELIBASE SKETCHER page 119 If necessary define geometric objects such as centroids see Defining Geometric Objects Section 10 2 page 138 Define the required geometric parameters i e the parameters you want to constrain These may involve just atoms or atoms and objects see Geometric Parameters Section 11 page 139 When you define each parameter constrain it as required e g specify a range of distances for a Relibase User Guide 71 6 5 6 6 72 bond length see Applying Constraints Section 12 page 144 Note if you wish to monitor a parameter rather than constrain it then you will normally have to edit the constraint in order to enlarge the allowed range to encompass that
161. face e g the hydrophobic portion The surface display is coupled to the pseudocentre display so we do this by turning off all the pseudocentres other than those that are hydrophobic e Use the pseudocentre tick boxes to turn off the display of non hydrophobic pseudocentres viz Donor Acceptor and Donor Acceptor You will need to click each tick box twice The first click will turn on all pseudocentres that are not already displayed the tick box will change from grey to white when this happens the second will hide all the pseudocentres of that type You are left with just the hydrophobic pseudocentres and the corresponding hydrophobic parts of the query cavity surface Explore non atomic graphics objects Right click items for available options Right click Item to search for similar query cavities 3 O0 Ca 0 O ee HOOW SkO O00880 vBase Hit pdb2jf0 9 in reli Cavity Surface mf Active Surface O Inactive Surface Unassigned Surface Pseudocentres Acceptor Pseudocentres Aliphatic Pseudocentres Aromatic Pseudocentres Donor Pseudocentres Donor Acceptor Pseudocentres Pi CavBase Query pdb1jus 5 in reli Cavity Surface M Active Surface Inactive Surface O Unassigned Surface Pseudocentres Acceptor Pseudocentres Aliphatic Pseudocentres Aromatic Pseudocentres Donor Pseudocentres DonorAcceptor Pseudocentres Pi e It is possible to
162. ficantly on going from the apo form of the enzyme 3app to the ligand bound form Relibase User Guide 1bxo e The reason for such a shift is quite obvious if the 1bxo ligand is now displayed back in the receptor You may wish to modify the display style and or the ligand s colour for clarity e Highlight any H bond interactions between ligand and receptor by activating the H bond tick box adjacent to Query in the Contacts panel of Hermes Note that if the Contacts panel is not present it can be launched via View Contacts At least two H bonds have obviously been formed as a direct consequence of this cavity collapse in the apo structure quite apart from a much reduced surface area exposed to solvent In fact if the default limits of the H bond contact are increased very slightly via the Define H bonds button more additional H bonds will be evident w Hermes J R File Edit Selection Display Yiew Calculate Descriptors Help Highlighting Depth Cueing Stereo ShowH HideH ShowUnk Hide Unk Graphics Objects Style wireframe Picking Mode Select Atoms v Clear Measurements f x x y y z z 90 4490 y 90 y 90 2 90 2 90 amp gt J T zoom zoom Atom selections Display Movable 4 Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1bxo 1 in reli Hit CavBase Hit pd
163. for user robertson Number of uervcaviy Cavi Search Date Comparisons T Ee ue e search pdbtjus 6 49 2 SS us May 10 17 15 search pdbtjus 6 49 pdb tjus 6 May 10 16 51 tutorial 1jus pdb1jus 5 May 9 15 52 search pdbidm 9 6 pdb1dms 9 Apr 26 15 40 e Clicking on any item in the Query Name column will show the hits from that particular search which you can then browse and view in 3D see Browsing the Results of a Search Section 4 6 page 113 e Clicking on an item in the Query Cavity column will take you to the cavity information page for that cavity see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 and will load the cavity into the 3D cavity viewer see Displaying and Comparing Cavities Section 3 page 101 Relibase User Guide 117 e Ticking an entry in the final column and then clicking on the Delete button will permanently remove the results of that search from your workspace Caution there is no undo facility or request for confirmation 5 Saving Cavities to File The molecular components ligands chains solvent molecules of a cavity or of an overlaid pair of query and hit cavities can be saved in mol2 format from Hermes Load the cavity see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 or the query hit pair see Browsing the Results of a Search Section 4 6 page 113 then select the top level menu option File followed by Save Cavity
164. from the CCDC S ftp server relibase data update package p ath_to_fi le Updates Relibase with the data update package specified by the path given in the package argument relibase data retry_failed If any entries failed to be applied to the Relibase database during a data update this command will attempt to re process them 188 Relibase User Guide relibase data delete database lt db gt Deletes from Relibase the entire database lt db gt using the database argument see Deleting a structure or a database Section 7 4 page 167 relibase data delete database entry entry to lt db gt _be_ deleted Deletes only the structure specified using the ent ry argument from the database lt db gt using the database argument see Deleting a structure or a database Section 7 4 page 167 relibase data_process input filena database conf conf_file me lt db gt mtz mtz_file template templ ate_file force Processes the single file filename and attempts to add it to the database lt db gt There are several optional arguments for this command conf allows you to specify a specific configuration file instead of the default mt z allows you to specify a MTZ file to be made available from the database entry template allows you to specify a template mol2 file to be used for a ligand in your input file This argument may be used multiple times
165. from the third pull down menu To specify a ring size range click on an atom with the right hand mouse button pick Cyclicity from the resulting pull down menu followed by Define Custom Ring Size then in the resulting pop up window select the required minimum and maximum ring size then hit OK closes window or Apply leaves window open The example below shows the setting required to specify that an atom must form part of a 5 6 or 7 membered ring Configure the smallest ring size Minimum ring size 5 atoms Maximum ring size 7 atoms Ok Apply Cancel Java Applet Window 5 Bond Properties 5 1 5 2 Changing the Current Bond Type The current bond type determines the type of any new bond created when drawing The current setting is shown at the bottom of the Draw window CHO N S P F Cl Any Other C Ligand ps single v Ba Ra The current bond type may be changed by clicking on this button and selecting from the resulting pull down menu Alternatively the bond type can be changed via the sketcher right click on the canvas ensuring no bonds are selected select Set Bond Type and pick the appropriate bond type from the resultant pulldown menu Any means any covalent bond bonds of this type are displayed as a dashed line Setting Variable Bond Types Bonds in a substructure may be variable e g double or aromatic The current bond type see Changing the Current Bond Type Section 5 1 p
166. gand in the CSD by traversing each path of up to 10 atoms within the atomic graph At each atom in the path a standard hashing algorithm is then used to set 2 bits in a fingerprint of 2000 bits in length The first bit is derived from a hash code that accounts for elemental type of the current node and the and the path already traversed The second bit is derived from a hash code that only accounts for atom types traversed along the current path The Tanimoto coefficient is set to a default value of 0 3 During a similar ligand search only ligands Relibase User Guide with a Tanimoto coefficient relative to the reference ligand above this threshold value will be displayed e By default the most similar ligand i e the one at the top of the hitlist is displayed Other ligands can be displayed by clicking on the CSD refcode e g AABHTZ AACANTI10 e The search results can be ordered highest to lowest similarity or vice versa by clicking on the Similarity tab at the top of the list of similar ligands e A 2D diagram is provided and can be enlarged by clicking on the image e Further information can be accessed via the following tabs above the 2D diagram e Diagram the tabbed view that contains the 2D diagram and basic crystallographic information and is shown by default when the search results are loaded e Details this view provides more comprehensive textual information including the publication details and more comprehensive crystallogr
167. ghly useful facility is the ability to search on non bonded interactions between a ligand and a protein or between two proteins Such searches can be set up also involve water molecules These kind of searches are useful for e g e Finding particular types of interactions between proteins and ligands e g hydrogen bonds contacts to metals etc e Generating tables of nonbonded interaction geometries e Finding a particular arrangement of amino acid residues e g catalytic triad SER HIS ASP e Looking for water mediated ligand protein interactions Relibase User Guide 6 6 1 Basic Guide to Nonbonded Protein Ligand Interaction Searching Draw the required substructures see CHAPTER 5 USING THE RELIBASE SKETCHER page 119 Ensure that the MoleculeType is set correctly for each substructure see Setting Molecule Types Section 1 3 page 120 Make sure at least one of the substructures is of MoleculeType Ligand Make sure each substructure is used in the definition of at least one distance constraint Run the search see Running a Search Section 6 8 page 74 6 6 2 Basic Guide to Nonbonded Protein Protein Interaction Searching Draw the required substructures see CHAPTER 5 USING THE RELIBASE SKETCHER page 119 Ensure that the MoleculeType is set to Protein for each substructure see Setting Molecule Types Section 1 3 page 120 Make sure each substructure is used in the definition of at least one distance constraint see Appl
168. h the various visualiser options More information about Hermes can be found elsewhere The GUI of Relibase is designed to allow easy navigation through the available data This is realised via hyperlinks which can be presented for example as ligand diagrams Go from the protein page to the ligand page for the ligand bound to this protein by clicking on the ligand 2D Relibase User Guide diagram for lacj This links you through to the Ligand Information page where the ligand will be displayed in Astex Viewer Hermes is updated automatically if the Automatic Visualiser Updates check box is activated Header HYDROLASE CARBOXYLIC ESTERASE Title Unavailable Compound ACETYLCHOLINESTERASE E C 3 1 1 7 COMPLEXED WITH TACRINE PROC NAT ACAD SCIUSA 90 1993 9031 JLSUSSMAN M HAREL SILMAN TORPEDO CALIFORNICA XRAY DIFFRACTION Cell a 113 700 b 113 700 c 138 100 alpha 90 000 bets 90 000 gamma 120 000 Spacegroup P3121 2 800 19 5 Deposition Date 18 8 1993 fz THA in PDB Entry 1acj Water Mediated Protein Ligand Contacts O TRP 84 O02 ASP 72 on ea seee Protein Chains pdblacj Show sequence Submit Search for Similar Chains haoo 8 Minimum Sequence Identity ficoo 8 Maximum Sequence Identity a Save in Hitlist I Overwrite Existing Hitlist Show Ligands Water information avBase Cavity Information e Use the Contacts functionality in Hermes to see if the ligand forms any indirect hydrog
169. hain centre movement in each case Below this list are tabulated details of the significantly different torsions that have been identified 92 Relibase User Guide Protein chain PDB 1gqr A Movement of side chain centers Residue Distance PHE 330 2 340 HIS 440 1 148 Changes in torsion angles Residue Torsion Atoms Reference Torsion New Torsion Difference TRP 64 CA CB CG CD1 98 178 112 135 13 957 PHE 330 C CA CB CG 31 387 107 844 76 457 PHE 330 CA CB CG CD1 61 773 117 354 55 581 HIS 440 C CA CB CG 61 866 0 285 61 580 TYR 442 C CA CB CG 138 699 149 446 10 747 e The header of the fourth column links to a summary table of sidechain movements for all chains in the Analysis Table Movements of greater than 1 5 are highlighted in red Sidechain Movements Reference chain PDB 1acj Reference ligand THA_999_pdb1acj_1 Note values above a threshold of 1 54 are highlighted in red Protein Chain PHE330 HIS440 PDB 1666 A PDB 1ea5 A 2 476 PDB 1gpk A 1 183 PDB 1gqr A 2 340 1 148 PDB 1h22 A 2 249 PDB 1h23 A 2 299 9 4 5 The Analysis Table Mutations and Insertions e The fifth column tabulates the total number of mutations insertions that occur between the reference chain and the superimposed chain As before information is only tabulated for those regions defined by the user i e either the whole protein or the binding site as defined by radius from the ligand The column
170. hand frame contains the Relibase protein entry page see Relibase Protein Entry Pages Section 4 1 page 27 for the currently selected entry from the list by default this will be the first hit Entries can be selected and inspected by clicking on the protein entry codes in the top left frame The following example shows the results for an author search for bode Relibase User Guide Relibaset wen f view Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Hits 1 100 of 183 gt gt pdb1a0l pdb1a5h pdb1a5i pdb1aoc pdbtast pdbiatl pdb1aus pdb1aut pdbtavg pdblavg pdb1azw pdbibda pdbibke pdbibqb pdbibqq pdbibqy pdbibth pdbibui pdbibuv pdbicst pdbicew pdbicgh pdbiclyv pdbiclyv pdbicsb pdbicse dbicse 183 hits for query bode Export XML Hitlist Save in Hitlist Browse Hit Headers Keyword Search Results Relibase 3 0 0 server rc2 gt View PDB Header Save PDB File PDB Website Bookmark Protein Entry 1a0l y Ligands Chains Solvent fal Clim v Schematic E J 1 Show Embedded Visualiser M Width 400 Height 300 Apply Hermes Controller Show in Hermes Automatic Visualiser Updates I Header SERINE PROTEINASE Title HUMAN BETA TRYPTASE A RING LIKE TETRAMER WITH ACTIVE SITES FACING A CENTRAL PORE Compound MOL
171. hand mouse button with the keyboard Ctrl key pressed down 102 Relibase User Guide The contents of the display area can be scaled i e zoomed in or out by moving the cursor up and down in the display area while keeping the right hand mouse button pressed down 3 2 2 Controlling Cavity Separation e Ifyou load a hit cavity from a similarity search it will by default be superimposed on the query cavity The default behaviour is for the surface areas of the query and hit cavities to only be displayed where they match The corresponding pseudocentres will be also be displayed and superimposed The spherical pseudocentres arise from the query whereas the tetrahedral pseudocentres arise from the hit cavity 3 2 3 Displaying Matching and Non matching Pseudocentres e Itis possible to change which pseudocentres are displayed for hit and query cavities via the Query and Hit drop down menus in the Cavity Controls window Options in the upper menu are to display All PC s in cavity PC s searched for Matched PC s default and Unmatched PC s Only the last two options are avaliable in the lower menu The two cavities can be moved apart by using the Separate cavities slider bar at the bottom of the Cavity Controls pane If you separate the cavities you will probably need to zoom out as well see Rotating Translating and Scaling Section 3 2 1 page 102 to keep everything in view x File Edit Selection Display View Calculate
172. heir sequence identity relative to the reference chain Now click on the Smiles Search button in the Relibase menubar Type C into the Enter SMILES Code box which will allow us to find all organic ligands and save Relibase User Guide 23 the results of this search in hitlist organic A molecular weight restriction could also be applied to remove small organic ligands such as acetate The same search could easily be carried out using the 2D 3D sketcher as described before and the results saved in a hitlist Hit the Submit button to start the search e Click on the Hitlists button on the Relibase menubar We want to be able to combine the two hitlists that have been created however it s not possible to combine a ligand type hitlist with a protein type hitlist so one of the hitlists must be converted into another hitlist type In this example the task was to find ligands so we will the convert the protein type hitlist MMP to a ligand type hitlist mmp_ligs e Select the hitlist you wish to convert using the pull down menu below Protein Set 1 e Select gt Ligand from the pull down menu of logical operators Enter the name of the new ligand set in the text box below New Set mmp_ligs Hit the Submit button adjacent to create the new hitlist Now you have list of all ligands contained in MMP 3 Combine Convert and Upload Hitlists for User susan Ligand Set 1 Operation Ligand Set 2 New Set Select existing hitlist AND x Se
173. his list In the resulting dialogue box enter the required Lower Limit and Upper Limit values In the example below a distance D has been constrained to values between 2 0 and 3 5A Label D1 t Lower Limit l2 ol Upper Limit 3 5 OK Cancel Java Applet Window In the case of torsions the chosen limits can be constrained to be within the range 0 to 360 degrees or 80 to 180 degrees Use the Change Torsion Range check box to change the convention used The same range must be used for all torsion angles in a search The Change Torsion Range box is inactive if more than one torsion angle has been defined Relibase User Guide 12 2 Crystallographic Constraints e Crystallographic constraints can be used to constrain the properties of atom s when setting up 2D or 3D queries in the Draw window e To constrain an atom right click on the atom and select Crystallographic Constraints from the resulting menu this will launch the Crystallographic Constraints dialog box Crystallographic Constraints B Factor oo upto 10000 Occupancy oo upto fo OK Cancel The range for the allowed values of crystallographic B factor and occupancy can be set 12 3 Water Descriptor Constraints e Water descriptors see Water Molecule Descriptors Section 5 6 page 4 can be used to constrain the properties of water molecules when setting up 3D queries in the Draw window e To constrain a water m
174. hrough the examples provided The first few tasks have been designed to introduce the basic types of information present in Relibase and the interaction of the GUI with the visualiser used in Relibase Hermes 1 2 Coverage of the Relibase Database e The database behind Relibase is the Protein Data Bank PDB http www rcsb org pdb It covers all entries in the PDB with the exception of theoretical structures However structures where a ligand substrate molecule was modelled into an experimental protein structure are included You can also use Relibase to search your inhouse database alongside the PDB e It is important to note that in Relibase all non protein moieties in a structure are considered to be either ligands or water molecules Hence metal ions anions solvate molecules cofactors and inhibitors are all regarded as ligands In the 3D visualiser DNA and RNA strands are displayed as ligands but they are ignored in ligand substructure searches e Each protein entry in the Relibase database corresponds to an entry in the PDB and contains the following information e Bibliographic textual and numerical information e Crystal structure data for X ray structures e Protein chain s e Binding site s e Chemical diagram of the ligand s e Crystal packing of the protein ligand binding site e Information about the water content of a particular protein Information about any cavities present within a particular prot
175. icates the total number of connected heavy atoms e g X4 indicates that the atom must be connected to 4 non hydrogen atoms e The letter a indicates acyclic c indicates cyclic Any further ring size constraints are indicated in square brackets e g c lt 7 indicates the atom must form part of a ring with a maximum ring size of no more than 6 members e Ifan atom is surrounded by a circle it is close to or on top of another atom Change to Select mode select the atom and move it away e Atom labels for carbon atoms may be hidden by selecting the top level menu Options and checking the Hide Carbons box e The font size used for labels can be changed by selecting Font from the View menu and picking the appropriate Increase Decrease Default font size 134 Relibase User Guide 8 Moving Scaling Rotating and Duplicating Substructures 8 1 8 2 Moving Atoms Select see Selecting Atoms Section 2 7 page 123 the atom s to be moved Press the left hand mouse button and move the cursor while keeping the button depressed Release the left hand mouse button at the desired position To move the complete query i e the entire contents of the sketcher window use the scroll buttons in the View Controls section to the left of the Draw window View Controls To re centre the complete query in the sketcher window select View from the top level menu followed by Recentre View from the resulting pull down menu To automatically move
176. ick on the Show in Hermes button e If no atoms were selected then the only options available are the Submit button to start the search or Cancel if you want to return to the drawing area e The progress of the search is displayed in the Messages box below the drawing area Clicking on any of the hits displayed in the Hits box while the search is progressing will open a new browser displaying the ligand entry page of the selected hit e To interrupt a search click on the Interrupt Query button e Hits are loaded in a second browser window and displayed as a browsable list of ligands see Using the Ligand Browser Section 3 3 page 22 If you interrupted the search all hits found thus far will be shown 6 8 3 Options Available on Search Hit Limits in Substructure Searches Relibase User Guide 77 stmt Semen AA AA A A Fitters Hitlist Controls Hit Limits Number of Hits Options Show maximum of 1000 hits D Show all hits Superposition file generation Batch search Run batch search with name Start Cancel The maximum number of hits can limited to a user defined number Enter the required number in the Show maximum of hits box e Alternatively all hits will be returned if the Show all hits radio button is selected 7 Similar Ligand Searches e Similar ligand searches can be carried out on ligands stored in the Relibase database i e ligands from the PDB see Sear
177. ict the molecular weight and size of ligands that you wish to retrieve from your search Leaving these boxes empty means that all ligands are considered e Lowest Resolution and Highest Resolution boxes can be used to restrict the experimental precision of the structures that you wish to consider Leaving these boxes empty means that all structures are considered If you only wish to consider structures with a resolution of 2 0A or better then enter 2 0 into the Highest Resolution box The resolution of NMR structures is set to 1 0A by default in Relibase so entering 0 0 into the Lowest Resolution box will exclude all NMR structures from the search Structure Method Filters the X ray and NMR check boxes can be used to filter searches based on the structure determination method e Use Hitlist allows you to restrict a search to a previously saved hitlist which can be selected from the pull down menu next to Use Hitlist Use Hitlists will only be available if you have carried out a search and saved the hitlist e Save in Hitlist allows you to save the results of a search in a hitlist type the required hitlist name into the Save in Hitlist box before you start the search Previously saved searches can be overwritten by typing the appropriate search name into the Save in Hitlist box and activating the Overwrite Existing Hitlist check box otherwise you will be prompted to give the search an alternative name e Use Databases allows you to sel
178. ified in a separate browser window Relibase User Guide Last Modification Mon Aug 11 2008 15 06 28 ue Aug 12 2008 13 51 47 Wed Aug 13 2008 15 06 55 Mon Aug 18 2008 15 02 12 Mon Aug 18 2008 15 50 20 Content ASCII XML ASCII XML ASCII XML ASCII XML ASCII XML Note any saved XML hitlist can be read back into Relibase see Loading XML Format Hitlists Section 6 8 page 55 e To edit the contents of a hitlist click on the name of the hitlist e g hydrolase The hitlist entries 52 will be displayed in the right hand frame Hits 1 40 of 10591 Protein Set hydrolase Add to Set esterase Submit Remove from Set hydrolase Submit Make New Set Submit I pdb2e6m I pdbtuih I pdb2pv9 F pdb1kgu I pdb2gv7 I pdb2i65 T pdb2ixt T pdb2qkb T pdb 1woj I pdb1xm4 I pdb2man I pdb1ug8 in ssetest T pdb1p3e T pdb1g2p T pdb1gd2 I pdb1c1p I pdb2o6m T pdb2gk6 I pdb2ane T pdbinym T pdb1p11 I pdb2eat l pdbitie I pdb2i8l in ssetest I pdb2iar T pdb1six e The above example shows protein hitlist entries for ligand hitlists the 2D ligand chemical diagrams will also be displayed Output options are also available for ligand hitlists see Saving Hitlists Section 6 6 page 54 e Click on the View Entries button to re load the entire hitlist or select a hitlist entry to link to the corresponding Relibase protein entry or ligand page depending on the hitlist type e Use the check boxes to select hitlist entries Selected
179. igand Unsaturated Rings and then Naphthalene The group is loaded by clicking in the drawing area Click Search and type naphthyl into the Save search in hitlist named box in the Start Search window Click on Start to run the search Combine the hitlists generated as described earlier recreating NAPAM again and ensure that the same number of hits are retrieved for each hitlist are the same using the different substructure searching methods 2 2 2 Combining Search Methods This search is also about combining various search methods The aim of this example is to try and find all organic ligands of MMP 3 stromelysin 1 which is a matrix metallo protease containing a zinc atom in its active site To do this select all ligands bound to MMP 3 sharing 100 sequence identity with stromelysin 1 PDB entry lums This will demonstrate the ability of the hitlist manager to translate ligand type hitlists into protein type hitlists Click on the Text Search button in the Relibase menubar Type lums into the Search String box and ensure the Search Type menu is set to PDB Entry Code Search Hit the Submit button to start the search Scroll down to the bottom of the Protein Information page retrieved for Lums to Search for Similar chains Save the results of the similar chain search in a hitlist mmp and then select the Submit button next to Search for Similar Chains to start the search The results are displayed as a table of chains ranked according to t
180. imum Chain Length restricts the chain length to the value equal to or above that entered in the text box from your current selection e Minimum Alignment restricts the alignment that is required between the reference and superimposed chains to be equal or above a certain value e By default all conserved residues in the chains are used initially for superposition then 40 are removed for the final superposition of hits The remaining 60 of residues are designated the Core residues If you want to use these 60 of residues for the superposition then click on the Use Entire Protein check box The superposition algorithm uses only the alpha carbon of each residue to carry out the superposition e If on the other hand you only want to use the binding site residues from the chain in order to carry out the chain superposition make sure that the For Superposition Use Binding Site Residues Only check box is selected Again it is the residue alpha carbons that are used in the superposition e If you want to look at the crystal packing for any of these similar binding sites then ensure that the Get Crystallographic Environment check box is selected Packing buttons will then be present in the Protein Explorer section of the Visualiser please refer to the Hermes documentation for further information e Ifrequired adjust the radius of the sphere around the ligand by entering a new value into the Radius of Sphere Around Ligand text box at the to
181. ing in CavBase see Cavity Similarity Searching Section 4 page 107 aims to find cavities or sub cavities in the database or databases if you have an in house database that match well with a query The query must itself be a cavity or sub cavity from the database Note only proteins that have cavities will be searched not the entire Relibase database e The similarity search program employs a clique detection algorithm described by Schmitt et al see References page 172 which treats the pseudocentres of the two cavities being compared as nodes of a graph The algorithm effectively matches some or all pseudocentres from the query onto similar pseudocentres in the hit The cavities are then superimposed by least squares fitting of the pseudocentres associated with the clique solution Pseudocentres with short pairwise distances and matching chemical properties are extracted and a simple function that estimates the overlap of the respective surface patches is used for calculating a similarity score value e There is a choice of scoring functions Scoring function 1 is the original scoring function as described by Schmitt et al see References page 172 Scoring functions 2 and 3 are modifications of the original function developed at CCDC specifically 2 and 3 differ in the way that the overlap in surface points are calculated In the original function the degree of mutual overlap is expressed by the number of surface points from pseudocenters
182. ings in Hermes click on the Show hyperlink adjacent to the appropriate cluster ring in the table after ensuring that the full complex structure has been first loaded into Hermes via the View in Hermes button 4 4 2 Water Mediated Protein Ligand Contacts Information concerning the number of water mediated protein ligand contact paths is given under the hyperlinkable 2D ligand diagram on the protein entry page 32 Relibase User Guide Ligands T16 in PDB Entry 1a3e Water Mediated Protein Ligand Contact Paths 2 GDF in PDB Entry 1a3e Water Mediated Protein Ligand Contact Paths 5 e Clicking on the 2D ligand diagram of e g the ligand with 5 water mediated protein ligand contact paths provides the following information on the ligand information page Ligands GDFEEIPEEYL in pdb1a3e Water Mediated Protein Ligand Contacts Show in Hermes N ILE 82 H NH1 ARG 67 H O GDFEEIPEEYL NH2 ARG 67 H O THR 74 H OE2 GDFEEIPEEYL 85 O TYR 76 H OH TYR 76 H OH GDFEEIPEEYL 156 70 4 GLU 80 H OE1 GDFEEIPEEYL OE2 GDFEEIPEEYL 182 O TYR 76 H e Clicking on any of the hyperlinkable mediating water molecules will lead you through to water descriptor information see Water Molecule Descriptors Section 5 6 page 4 e In order to highlight certain water mediated protein ligand contacts in the embedded visualiser activate the relevant tick box under the column headed
183. ish to remove the query cavity s H atoms as before to simplify the display It is also useful to be able to rescale these structures by moving the mouse vertically with the right mouse button depressed e When this is done you will notice that these two cavities are very closely matched e The display of protein chains ligands and solvent can be controlled by deactivating the relevant tickboxes in the Protein Explorer Relibase User Guide 49 Also the display of pseudocentre Surfaces can be controlled by toggling the relevant Surface boxes in the Graphics Objects Explorer Hide the protein and solvent atoms but keep the matched surfaces displayed Now use the slider in the Display Controls panel of the Cavity Controls window to separate the two cavities Given the 100 homology of the protein structures and the very close similarity between the two ligand structures it is not very surprising that the matched parts of the cavities are virtually identical It is usually more informative to compare that part of the hit surface that matches the query with the full query surface To do this with the mouse still in the Cavity Controls panel click the pull down on the Show Pseudocentres Query line that shows pseudocentres and select PCs searched for In this particular instance where 35 matches have been made out of a possible 36 there is still almost no discernable difference The easiest way to find the single missed pseudocentre is to turn
184. ist AND Setectewstngnitist le gt Submit ee Protein Set 1 Operation Protein Set 2 New Set T pdb2zin 9 M pdb1vxo 3 Select existing hitist AND Selectexistinghitist gt Submit I pdb1ax9 2 I pdb1b65 19 3 Cavity Set 1 Operation Cavity Set 2 New Set Cpdbiwiz 4 I pdbdiar 1 Select esting nitist AND Select existing niist E gt Submit a Database 1 Operation Set2 New Set Mpdbirqu4 F pdb2ow 3 I pdb1soj 2 I pdb1zgb 3 MINUS z Select existing hitlist z gt Submit Cpababx2 Co pdb1mx5 9 Codhinnna I ndh avn S E All databases Ei e From the resulting list of cavities select the hyperlink next to the check box for the cavity you wish to view 3 Displaying and Comparing Cavities 3 1 Displaying a Cavity e A view of the cavity opens automatically in Hermes whenever you click on a cavity hyperlink e g the text pdbla4g 2 in the display below pdb1a4g 2 Further details of how to access cavity information are given elsewhere see Accessing Cavity Information for Relibase Database Entries Section 2 page 99 Relibase User Guide 101 e The protein around the cavity and any ligands bound will be displayed in the visualiser The cavity itself will appear as a solid surface which is coloured according to the aadjacent pseudocentre types The correspoding pseudocentres are also displayed In addition a Cavity Controls window will appear Hermes File Edit Selection Display View Calc
185. ituations Find proteins containing two indole groups of tryptophan residues in close contact Investigate one of the resulting hits while the search is still running Describe the Trp Trp interaction geometry of one of the hits 28 Click on the Sketcher button on the Relibase menubar Click on the Other button in the Templates part of the Relibase interface to the left of the drawing area then select Protein and Tryptophan from the resultant pull down menus Load the template by clicking to the left of the drawing area Define the centroid of the indole ring by clicking on the Add 3d button and selecting all the atoms that make up the ring 9 in total This can be done more easily by depressing the left mouse button and dragging a rectangle which incorporates all nine ring atoms Once all have been selected click Define next to Centroid in the Add 3d pop up window The text CENT will be given in the Defined objects section of the Add 3d window Click Done once you have finished Load a second tryptophan template in the same manner ensuring it is loaded to the right of the drawing area Define the centroid of the indole ring in the same way as outlined before but keep the Add 3d window open Set up a distance constraint left click on CENT and CENT2 Once both CENTI and CENT2 are selected click on the Define button next to Distance Relibase User Guide Relibase Smiles Search Sketcher Hitlists Stored Results 2D 3
186. ity map viewed as a contour surface gives a good 3D representation of the shape and internal structure of the molecules dependent on both the resolution and degree of error in the experimental diffraction measurements and their processing and is used to build the atomic model of the structure A number of forms of the electron density map can be calculated to highlight different types of information The two most commonly displayed are the 2Fo Fc and Fo Fc maps e The 2Fo Fc map blue provides a representation of how well the model agrees with the experimentally derived electron density distribution The Fo Fc difference map specifically highlights areas of disagreement between the two Positive green density indicates regions where structure is present but has not been modelled and negative red density indicates regions where atoms have been modelled that are not supported by the underlying experimental data In both cases the detail visible in the maps will be dependent on the resolution of the experimental data and may contain artefacts due to errors in measurement or processing of the data 184 Relibase User Guide APPENDIX D Configuring the Ligand Diagram Generation In order to generate 2D diagrams for in house structures you will need to use 3rd party software Relibase can make use of e Tripos SYBYL Note that versions of SYBYL prior to 7 0 are not compatible e ChemAxon MarvinBeans Please note that OpenBabel is also required
187. k Relibase User Guide 65 66 File Edit View Atom Bond Options Query Sketcher 2D 3D Sketcher retibase 3 0 0 res Number of connections to non hydrogen atoms Cyclicity Select Fragment Place Fragment in Ligand Delete Atom Type Protein Help 3d Parameters D1 Options Delete ele e With the cursor on any one of the phenylalanine atoms see above click the right hand mouse button and from the resulting drop down menu select Secondary Structure Constraints The following panel will appear Relibase User Guide Secondary Structure Constraints Helices Sheets amp Strands Turns Helix Type Y Ignore Helix Type Right Handed Helices l Allow Alpha Helix _ Allow 310 Helix C Allow Pi Helix C Altow Gamma Helix C Allow Omega helix Handed Helices C Allow Alpha Helix Allow Gamma Helix _ Allow Omega Helix Other 27 ribbon helix C Potyprotine C Must not be in a helix Helix Length Minimum Length oH Maximumtength 1 000 4 Z Use original PDB helix assignments Reset Constraints Helices Sheets amp Strands Turns ok cancer e You have already identified that the DFG substructure you wish to find is not a helix but rather part of an open 4 residue type VII3 turn and so the default window shown above is not appropriate e Click on the Turns tab and then se
188. lay of multiple structures Hermes see Hermes Section 5 2 page 47 is provided to facilitate more detailed investigation of the hit structures Astex Viewer Astex Viewer is a Java molecular graphics program developed and distributed by Astex Therapeutics http www astex therapeutics com Basic functionality is documented below Advanced functionality is available by right clicking More detailed information can be found in the Astex Viewer documentation http www astex therapeutics com Astex Viewer Astex Viewer2 doc interface html This documentation is also provided in the Relibase distribution e Rotation move the cursor around in the 3D window while keeping the left hand mouse button pressed down Translation move the cursor left or right in the 3D window while keeping the left hand mouse button and the Control key pressed down e Scale zoom in or out by moving the cursor up and down in the in the 3D window while keeping the left hand mouse button and the Shift key pressed down e The appearance of the 3D display varies depending on the type of page 42 e Protein information page see Appearance of AstexViewer on a Protein Information Page Relibase User Guide Section 5 1 1 page 43 e Ligand information page see Appearance of AstexViewer on a Ligand Information Page Section 5 1 2 page 44 e Binding site information page see Appearance of AstexViewer on a Binding Site Superposition Page Section 5 1 3
189. ld by looking at correlations between raw scores e Prepare a plot of the descending Normalised Score for all 400 saved cavity matches e It should look something like this albeit without the coloured highlighted features Relibase User Guide 1bx0_36 Normalised Score ScoreFunction_3 High scoring cluster of 11 cavities 100 iomologous with the quer Mid range matches in descending score transition point tore a lu Cluster of meaningless random rinata cavity matches Da lt l ni aon a pe SEpesnReener aS nee sais Bae ieee 3 z fs 55 555 55SS5S54S55 gt S55 gt HER 8 ae zz S presiecisiisis seestarestseettcpesessestsescy E aes Ssshssse ssse E Sss see Sire e PFPA F El STS SS 3 SE EEE EERIE E SSS CREE EELS E PESEERRRREE EN zz SSS eee EN co 5 a E 8 4 Be e Inspection of this plot would suggest that the 400 saved cavity matches fall broadly into three distinct groups e A high scoring cluster Normalised Scores between 97 5 74 4 of 11 receptors completely homologous with pdb1bxo e A large group of intermediate level matches 195 cavities with Normalised Scores between 69 27 e A residual group of approximately equal size of meaningless random cavity matches e You will also notice that in the body of the mid range cluster are two further cavities that are c
190. lect existing hitlist gt Submit Protein Set 1 Operation Protein Set 2 New Set MMP 7 gt Ligand gt mmp_ligs Read XML format Hitlist _Browse Submit e Combine the two hitlists MMP_LIGS and ORGANIC using the AND operator to create a ligand hitlist orgmmpligs which correspond to the task required Inspect the list to check that the structures expected have been found so far 2 3 3D Interaction Searches The following examples are designed to explore the abilities of the sketcher for setting up 3D searches e g for particular protein ligand interaction patterns The sketcher supports three types of molecules protein ligand and water for specifying the respective types of fragments Combinations of these types are also available e g Protein or Ligand Protein or Water Ligand or Water in addition to an Any molecule type Independent fragments must be correlated by applying constraints 1 e distance angle or torsion angle constraints 2 3 1 Protein Ligand Interaction Searching Use the AMIDINO hitlist generated in the previous example and search for the ligands which form a salt bridge with a carboxylate group of a protein Use distance constraints to find bidentate salt 24 Relibase User Guide bridges e Click on the Sketcher button on the Relibase menubar to take you into the Relibase sketcher e Draw the amidino group attached to a carbon atom as in the previous example but this time use Any bond type fo
191. lect the 4 Residues tab Now select the open tab and toggle off the Ignore Turn Type check box e Activate the tick box adjacent to turn type VII3 e Because our DFG substructure is so heavily conserved although not in fact the initial Ala residue it is likely that the position of each residue in the fragment is also important and you should therefore toggle off the Ignore Position check box and check on the Third position The Secondary Structure Constraints window should now look as below Relibase User Guide 67 68 Secondary Structure Constraints Helices Sheets amp Strands Turns 2Residues 3Residues 4Residues 5Residues 6 Residues normal open reverse Turn Type _ Ignore Turn Type r _ Vib Jye Villa Villb Ex La Xl XII XIII Viltb J2 A3 Vil4a _ Vil4b __ Must not be one of selected turn types open B turn Position _ Ignore Position _ First _ Second v Third _ Last Reset ee Reset Constraints Helices nj Sheets amp Strands Turns Ok Cancel Press Ok to return to the sketcher Click the Search button to the left of the sketcher window to start the search For this particular job we do not wish to search for NMR or DNA structures so deactivate the check boxes adjacent to these options And in this specific example prior inspection of the site
192. lected by hitting Edit in the top level menu and Select All in the resulting pull down menu or by using the CTRL A shortcut In any mode the current selection can be reversed by hitting Edit in the top level menu and Invert Selection in the resulting pull down menu or by using the CTRL I shortcut Everything that was selected becomes unselected and vice versa In any mode everything can be deselected by hitting Edit in the top level menu and Deselect All in the resulting pull down menu keyboard shortcut CTRL SHIFT A In any mode a bonded fragment may be selected by right clicking on an atom or bond in the Relibase User Guide fragment and selecting Select Fragment 2 8 Deleting Atoms and Bonds There are several methods including e In Delete mode click with the left hand mouse button on the atom or bond to be deleted e In any mode click with the right hand mouse button on the atom or bond to be deleted and pick Delete Atom or Delete Bond from the resulting pull down menu e Select the atoms and bonds that you wish to delete see Selecting Atoms Section 2 7 page 123 then click with the right hand mouse button on a blank point in the white area and pick Delete Selected from the resulting pull down menu or by using the keyboard Delete shortcut e To delete all atoms and bonds in any mode move the cursor onto a blank point in the white area click on the right hand mouse button and pick Delete All from the pulldown menu
193. losely with the binding site Note one of the fragments has to be a ligand Also it will be necessary for a 3D constraint to Relibase User Guide 75 76 be set up between the two fragments in order for Relibase to carry out the search This constraint can be set to be loose however if needed If the Search packing environment box is checked then the Only packing check box becomes activated Checking this box means that the search will only consider cases where the second protein or ligand fragment is part of a neighbouring protein packed closely with the binding site If the query contains two protein fragments then the Allow intra chain contacts check box becomes activated By default this check box is toggled on If however it is desired to search only for contacts between two protein chains separately identified in the pdb entry then this box should be toggled off If the query contains two ligand fragments then the Allow intra ligand contacts check box becomes activated The default behaviour is for contacts to be found only between two different ligands within the same pdb entry Toggle this box if you wish also to consider intra ligand contacts If you want to create an overlay of the hits you must select at least three or more non hydrogen atoms you wish to superimpose before starting the search Then in the superposition file generation section of the Start Search dialogue box toggle on the Superimpose hits on selected atoms
194. lting pull down menu followed by Define Custom Ring Size then in the resulting pop up window select the required minimum and maximum ring size then hit OK closes window or Apply leaves window open The example below shows the setting required to specify that a bond must form part of a 5 6 or 7 membered ring Configure the smallest ring size Minimum ring size l5 atoms sen Maximum ring size 7 atoms Apply Cancel Java Applet Window 6 Using Substructure Templates Substructure drawing can be made easier by using templates which are pre drawn substructural fragments e To access the available templates hit the Other button in the Template sections on the left of the Draw window then select either Protein or Ligand templates in the resulting pull down menu e Templates are available for ligands and for proteins e For ligands amino acid steroid and saturated and unsaturated ring templates can be accessed e For proteins standard amino acid templates as well as modified amino acid templates e g phosphorylated tyrosine are provided e Once selected the template should be moved into the desired position using the mouse To scale the template move the mouse while keeping the Shift button depressed to rotate move the mouse while keeping the Control button depressed To fuse the template with an existing substructure of the same molecule type move it towards the substructure Fusion is achieved by overlappi
195. ly be shown a table summarising the search results If you exit Relibase while the search is in progress you can use the search management tool to access the same table see Managing Search Results Section 4 7 page 116 e A summary of the search settings is given at the top of the Cavity Comparison Search Result window followed by a table of search results The results table will look something like this Relibase User Guide 113 Search Results 1 15 of 400 TE O q Oo 114 Select Cavity Score Normalised Matched Protein Cavity Header Score Centres Homology Homology ACID PROTEINASE PENICILLOPEPSIN COMPLEX i es FYDROLASE WITH PHOSPHONATE INHIBITOR CRYSTALLOGRAPHIC ANALYSIS OF HYDROLASE ACID TRANSITION STATE MIMICS BOUND TO PROTEINASE PENICILLOPEPSIN PHOSPHORUS CONTAINING PEPTIDE ANALOGUES ACID PROTEINASE PENICILLOPEPSIN E C 3 4 23 20 COMPLEX WITH PHOSPHONATE INHIBITOR 100 0 90 2 HYDROLASE METHYL 28 1 N FORMYL L VALYLJAMINO 2 2 NAPHTHY LIETHYLJHYDROXY PHOSPHINYLOXY 3 PHENYLPROPANOATE SODIUM SALT CRYSTALLOGRAPHIC ANALYSIS OF HYDROLASE ACID TRANSITION STATE MIMICS BOUND TO PROTEINASE PENICILLOPEPSIN PHOSPHORUS CONTAINING PEPTIDE ANALOGUES CRYSTALLOGRAPHIC ANALYSIS OF TRANSITION STATE MIMICS BOUND TO PENICILLOPEPSIN PHOSPHOROUS CONTAINING PEPTIDE ANALOGUES 100 0 76 0 100 0 90 2 HYDROLASE ACID PROTEINASE 100 0 92 7 Each row of the table relates to a hit cavity f
196. ly to the corresponding Ligand Information page Relibase User Guide 13 Relibaset HALA View Keyword Search Results retibase 3 1 0 Hits 1 3 of 3 Result of a 2D Similarity Search for GDFEEIPEEYLQ_54 I_pdb1fph pdb1a5h Minimum Similarity 0 7 Submit Ta Hits 1 40 of 936 Hits 1 3 of 3 Similarity Index GDFEEIPEEYL DFEEIPEEYLQ 3 hits for query hydrolase Export XML Hitlist Save Search Results DFEEIPEEYLQ Browse Hit Headers The search results can be filtered on the basis of the Tanimoto coefficient the default value is 0 7 Enter the required minimum similarity index a value between 0 and 1 into the Minimum Similarity window and hit the Submit button e The hitlist of similar ligands can either output as an XML format file or saved on the Relibase server or using the Export XML Hitlist or Save in Hitlist buttons respectively 1 6 Similar Binding Site Searches Relibase allows superposition of similar ligand binding sites if the proteins share a significant level of sequence similarity This two step approach can be used to easily analyse the common features and differences between those binding sites such as protein flexibility water conservation ligand clashes etc Find ligands containing a 5 iodouracil moiety using text based searching 14 Relibase User Guide e Load the ligand page for one of the two binding sites in the PDB entry 1ki6 which is a thymidine kinase complex The starting p
197. m the Search Type drop down list and type esterase into the Search String box Enter a suitable name e g tutorial into the Save in Hitlist box Hit the Submit button to run the search e When the search is finished hit the top level Hitlists button There will be a TUTORIAL hitlist stored which we will use as a database subset for our cavity search Relibase User Guide 3 3 3 Setting Up the Search Query e Now we turn to the cavity similarity search The first thing is to find the query protein cavity e Hit the top level Text button and do a keyword search for QacR e There are several relevant structures of which we will use just one Click on pdb jus in the list of hits to show the Protein Information page for this structure Hit the Show in Hermes button located in the Hermes Controls Panel this will launch the Relibase visualiser As you see this is a structure of the QacR protein with a bound ligand rhodamine 6G Ty Hermes x File Edit Selection Display View Calculate Descriptors Help Highlighting W Depth Cueing Stereo ShowH HideH ShowUnk Hide Unk Graphics Objects Style Wireframe Picking Mode Select Atoms Clear Measurements x x y y z z x90 x 90 y 90 y 90 2 90 2 90 amp gt 4 T zoom zoom Atom selections e Go to the very bottom of the webpage and click on the Cavity Information button e You now see a list of the cavities in this protein structure We will use as a query
198. me robertson is given and the databases that are currently loaded for searching alongside each other In this case there are three inhouse databases battletwo ian and jase in addition to the PDB itself reli From this page in the interface it is possible to access the Data Processing Graphical User Interface GUI for generating inhouse databases In addition cavity hitlists generated using CavBase may also be viewed Most Relibase searches can be started from the following buttons on the Relibase menubar Text Search Allows searches on entry code text HEADER TEXT COMPND and SOURCE records author name ligand compound name and ligand entry code searches Sequence Search Allows you to search on amino acid sequence Smiles Search Allows you to retrieve ligand SMILES strings Sketcher The drawing area allows searches on 2D ligand substructure searching 3D ligand substructure searching and nonbonded protein ligand or protein protein interaction Hitlists The hitlist manager allows you to view and combine results from previous Relibase Relibase User Guide 1 4 searches e Stored Results View the results of saved searches e Help This links through to the Relibase Help pages which includes both the Relibase and Reliscript User Guides and the Relibase Technical Documentation Some Relibase searches can only be started from Protein Information pages or from Ligand Information pages these are e Ligand similari
199. mit Read XML format Hitlist Browse Submit Searches can also be restricted to a hitlist generated in a previous step See how many structures published by both Bode and Stubbs are hydrolase structures Click on the Text Search button in the Relibase menubar Select Keyword as the Search String and type hydrolase into the Keyword Search box ensuring the Search Field menu is set to HEADER TITLE COMPND and SOURCE Records Select your combined author hitlist name e g COMBINED from the Use Hitlist entry box then hit Submit Inspect the hits for example 1fph Relibase also allows searching for similar ligands using topological fingerprints These searches can be invoked from any Ligand Information page Find out if the ligand in the thrombin structure 1fph was ever used as a ligand in another structure in the PDB and all the ligands which are most closely related to this ligand From the GDF Ligand Information page of 1fph click on the Similar Ligands Search button at the top of the page All ligands in the Relibase database are compared to the reference ligand highlighted in red The results are loaded into the browser as a list of ligands and are ranked in decreasing order of similarity to the reference ligand The similarity index given in the first column is a Tanimoto coefficient and the 2D diagrams in the second column are linked to the corresponding ligand pages If the search results in only one hit then you will be linked direct
200. n Ligands in the Brookhaven Protein Databank M Hendlich F Rippmann G Barnickel Journal of Chemical Information and Computer Sciences 37 774 778 1997 Relibase e Use of Relibase for Retrieving Complex 3D Interaction Patterns Including Crystallographic Packing Effects A Bergner J Gunther M Hendlich G Klebe M Verdonk Biopolymers Nucleic Acid Sci 61 99 110 2002 e Relibase Design and Development of a Database for Comprehensive Analysis of Protein Ligand Interactions M Hendlich A Bergner J G nther G Klebe J Mol Biol 326 607 620 2003 Utilising Structural Knowledge in Drug Design Strategies Applications Using Relibase J G nther A Bergner M Hendlich G Klebe J Mol Biol 326 621 636 2003 Uppsala Electron Density Server e The Uppsala Electron Density Server G J Kleywegt M R Harris J Zou T C Taylor A Wahlby T A Jones Acta Cryst D60 2240 2249 2004 DOI 10 1107 S0907444904013253 http eds bmc uu se Water Information Module WaterBase e Cluster Analysis of Consensus Water Sites in Thrombin and Trypsin Shows Conservation 172 Relibase User Guide Between Serine Proteases and Contributions to Ligand Specificity P C Sanschagrin L A Kuhn Protein Sci 7 2054 2064 1998 Valence Screening of Water in Protein Crystals Reveals Potential Na Binding Sites M Nayal E Di Cera J Mol Biol 256 228 234 1996 Knowledge Based Scoring Function to Predict Protein Ligand Interac
201. n Search Results page were some additional options One of these is Superpose Selected Cavity Binding Sites In fact the default state is that all the structures are superimposed which is why you didn t have to use this option before running the previous part of the tutorial However on occasion you may wish to look at specific multiple superimposed cavities in Hermes without having to load the entire set of solutions which in this instance would make Hermes run slowly To superimpose selected hits we must first select the structures of interest by activating the tick box in the first column of the table Select 20ah 2 cavities 2web 1 cavity 2vj9 3 cavities and lepr 1 cavity then click on Superpose Selected Cavity Binding Sites To view the superimposed structures that we have selected click on the Display Superimposed Cavity Binding Sites in Hermes hyperlink This option offers you the possibility of inspecting the various binding sites and ligands after they have been superimposed according to a receptor based rationale There are a number of existing methods for superimposing ligands but not many that deal reasonably with dissimilar structures One of the objectives one might wish to accomplish with such an aligned set of structures is to use the set as a source of ideas for de novo hybrid compounds It is unfortunate that the subset of protein structures saved from your original cavity search contained a large number of meani
202. n only be selected to be saved after the search has been run e In order to specify that you would like to save a hitlist before running a search type the required hitlist name into the Save in Hitlist box The example below shows a protein hitlist called ESTERASE being saved for keyword search Relibase Pee View Home Text Search Sequence Search SMILES Search Stored Results Text Sea rch Relibase 3 0 0 server rc2 Search Type Keyword x Search String esterase Match whole words only I m Keyword Search Options Search Field HEADER TITLE COMPND and SOURCE Records m Filter Search Options Minimum MolWeighth SSCS Maximum MolvVeight a Highest Resolution A joo o Lowest Resolution A Structure Method Filters Xray M NMR M Use Hitlist Select existing hitlist _v Save in Hitlist esterase Overwrite Existing Hitlist gregtest reli Use Databases Ssetest Submit e To overwrite a previously saved hitlist type the hitlist name into the Save in Hitlist box then activate the Overwrite Existing Hitlist check box and click Submit e A hitlist can be saved after a search has been run by clicking on the Save in Hitlist button which Relibase User Guide 49 will be located in the bottom left frame of the results window for text sequence SMILES and 2D 3D searches or the top of the ligand similarity results page for ligand similarity searches 413 hits for qu
203. n sh make sure COOT is in the path PATH where ever coot bin SPATH create temporary directory mkdir tmp s dir ed tmp dir unpack tarball tar xf 1 pdb 1s pdb head n 1 mtz ls mtz head n 1 fire off coot coot pdb Spdb auto mtz exit and remove temporary directory Relibase User Guide cd tmp amp amp rm fr tmp dir exit 0 Note that if you want the Save PDB MTZ File button to produce a zip file instead of a tar file this can be configured in the RELIBASE_ROOT relibase_htdocs include global_settings php Change the default from tar to zip in the line define PDBMTZ_ARCHIVE_FORMAT tar zip or tar Relibase User Guide 181 182 Relibase User Guide APPENDIX C Electron Density Configuration and Viewing Electron Density Configuration e Relibase has the ability to store structure factors and to display electron density maps in the web based visualiser However Relibase does not have built in software for converting structure factors to electron density maps It therefore requires access to CCP4 Attp www ccp4 ac uk libraries and software If Relibase does not have access to CCP4 libraries and software it will work as normal but you will not have the ability to visualise electron density maps in the web based visualiser e During the installation you will be asked to optionally specify the location of your CCP4 libraries and software If
204. n the original PDB file A text based search using the regular expression bovine trypsin will not return all bovine trypsin structures since these two words separated by exactly one space are not guaranteed to be present in every PDB bovine trypsin entry However searching for bovine trypsin will bring up hits that contain these two words in any order and position and should contain all the relevant structures provided that both words everything in the search string occur in at least one of the PDB fields noted above 3 2 Author Name Searches Relibase User Guide 59 3 2 1 Performing an Author Search e Click on the Text Search button in the Relibase menubar e Select the Keyword option in the Search Type box e Select the Author Name option from the Search Field pull down menu e Type the required text string into the Search String box Relibase PDB Entry Code I Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Text Search relibase 3 0 0 rcs Search Type Keyword z Search String Harel Match whole words only I Use regular expressions Keyword Search Options Search Field Author Name 4 Filter Search Options Minimum Year Maximum Year Minimum MolWeight Maximum MolWeight Highest Resolution A 1 0 Lowest Resolution A Structure Method Filters Xray M NMR M Use Hitlist Select existing hitlist _v Save in Hitlist harel Overwrite Existing Hi
205. nalysis Table Conserved Waters Section 9 4 7 page 95 e Clashes with Proteins see The Analysis Table Clashes with Proteins Section 9 4 8 page 95 9 4 1 The Analysis Table Protein Chain e The first column presents the superimposing protein chain Each entry in this column links to a PDB entry page 9 4 2 The Analysis Table RMS e The second column gives the RMS figure for the entire chain on chain superposition RMS overall The RMS is calculated from the alpha carbons of each correctly aligned residue This is the RMS value that is given by default e Two additional RMS values can also be calculated by checking the RMS core and RMS binding site check boxes at the base of the table and then clicking on the Recalculate Table button again at the base of the table e The RMS core is the RMS calculated using only the alpha carbons of the Core residues The core residues are those residues selected for final superposition of hits after the initial superposition of all conserved residues is performed see Performing a Similar Binding Site Search Section 9 1 page 84 e The RMS binding site is the RMS calculated using the alpha carbons that make up the binding site defined using the option Radius of Sphere Around Ligand A in the similar binding sites superposition setup page default value is 6A see Performing a Similar Binding Site Search Section 9 1 page 84 9 4 3 The Analysis Table C Alpha Movements e The third column giv
206. nce pdb2pks C 102 Amino Acids Query Sequence 7 Amino Acids Identity 100 0 EGKRGDACEGDSGGPFVMKSPFNNRWYOMG IV SWGEGCDRDGKYGFYTHVFRLKKWIOQKV IVSWGEG This alignment has been carried out using FASTA 3 5 Sept 2006 optimized The percentage Identity shown on the Alignment of Entries page 100 0 above is for the entire hit protein compared to the query protein determined by ALIGN e The ligand diagrams in the listing of chains are only shown if Show Ligands was selected when the search was started see Protein Sequence Searches Section 4 page 65 Click on the diagrams to launch the individual ligand pages in a separate browser window see Relibase Ligand Pages Section 4 2 page 28 3 5 Viewing 3D Substructure Search Results Geometrical Analysis 3 5 1 Viewing Distribution Histograms for Geometrical Parameters e When you have defined geometrical parameters in your query see Geometric Parameters Section 11 page 139 histograms for these parameters are generated automatically e The histogram s can be viewed by clicking on the Histogram s link in the bottom left frame of the Relibase ligand browser see Using the Ligand Browser Section 3 3 page 22 1452 hits Histogram s e This loads the histogram s into the browser Relibase User Guide 25 Relibaset PDB Entry Code J Sequence Search SMILES Search Stored Results Histog FAaMS relibase 3 0 0 RCS Export Histogram Data as CSV Export Histogram Data as T
207. nction Bear in mind that some structures of a particular protein may suffer deletions due to poor resolution of one or other loop of residues So it may be necessary to set the lower limit of sequence identity to be less than 100 in order to collect all structures of a particular protein 9 Similar Binding Site Searches and Superposition The definition of a Similar Binding Site in the context of this section is a binding site which has a significant degree of homology with the reference binding site Similar binding sites in terms of surface shape and properties but which have low homology with the reference structure may also exist These can be sought using the Cavbase module see CHAPTER 4 RUNNING SIMILAR CAVITY SEARCHES page 97 You may wish to use a similar binding site search for some of the following reasons e To compare the binding modes of different ligands at a particular binding site e To compare the binding mode of one ligand in two closely related binding sites To find bioisosteric replacements e To analyse ligand induced fit and protein flexibility To find conserved and displaced water molecules 9 1 Performing a Similar Binding Site Search e From any ligand page click on the Similar Binding Sites Search button on the menu bar above the 2D diagram of the ligand 84 Relibase User Guide e A form is loaded into the browser Reference Ligand is THA in PDB Entry 1acj Chains Binding to Ligand Select Ref
208. nd relibase data_process input path to structures database md_snapshots conf path to md_processing conf Note that Relibase has hardcoded defaults for the data processing configuration options these are overridden by the flags set in the relibase_processing conf file When using an alternative configuration file the options are set in the following order 1 The hardcoded defaults get set 2 The relibase_processing conf options are parsed and set 3 Any options set in the alternative configuration file are parsed and set 8 5Deleting a structure or an in house database e In house structures and databases can be deleted from Relibase using the command below S relibase data delete database specification entry entry_to_be_deleted e So suppose you wanted to delete a database named aaa then you would use the command relibase data delete database aaa e Whereas if you wanted to delete an entry named snapshot1 from a database called md_snapshots you would use the command S relibase data delete database md_snapshots entry snapshotl Relibase User Guide 171 References ReLiBase e Databases for Protein Ligand Complexes M Hendlich Acta Crystallographica D54 1178 1182 1998 VODAK e Constituting a Receptor Ligand Information Base from Quality Enriched Data K Hemm K Aberer M Hendlich Intelligent Systems for Molecular Biology 170 179 1995 BALI e BALI Automatic Assignment of Bond and Atom Types for Protei
209. nd center on clicking link Turn Display lan Turns v Assignment Method SHAFT ell e These rows indicate that the specific geometric features experimentally observed with this residue come within the defining tolerances for three different turn types A certain amount of prior knowledge is therefore helpful when it comes to deciding which of these three turn types is most useful when it comes to solving our present problem Note the turn class describes all turns of similar length the turn type is the specific clustered turn geometry see the turn classification paper But we do know that this DFG sequence is totally conserved for all the Tyr Ser Thr kinases that are reported to be susceptible to this conformational switch and it might be reasonable to expect such a stable configuration to be part of a single structural fragment and therefore common enough to have been identified elsewhere We can also see from inspection of the 3D image that the next five residues form a well ordered helical substructure which again we might expect to see with a single known classification e The only classification for Phe382 consistent with this information is as member of an open 4 residue type VIIS turn according to the turn classification The shorthand version for this provided in the table cell 0 4 VII3 turn Click the cyan coloured cell marked as this type under the Phe A382 heading The side chains will appear highlighted in ball and
210. nd diagrams to be displayed in the resulting table of chains make sure the Show Ligands check box is selected e Ifyou wish all chains included in the 3D superposition to be preselected in the list of results ensure that the Preselect Protein Chains check box is switched on this is the default otherwise switch off this check box and then make your selection from the list of results Relibase User Guide 15 16 Relibaset Keyword Search Results relibase 2 1 0 Hits 1 7 of 7 Superposition and Analysis of Similar Binding Sites Ligand Induced Flexibility Conserved Water Molecules in Contact with Ligand Reference Ligand is AHU in PDB Entry 1ki6 Chains Binding to Ligand Select Reference pdbiki6 A Residues 300 Protein Chain Parameters Minimum Sequence Identity 99 0 0 Maximum Sequence identity 100 0 j Search Parameters yN Lowest Resolution A OOS S S Wd Options Preselect Protein Chains M Show Ligands Submit j Manfred Hendich 1994 1999 and Cambridge Crystaliographic Dalta Centre 7999 2006 7 hits for query Start the search for similar chains by clicking on the Submit button The results are displayed as a table of chains ranked according to their sequence identity relative to the reference chain Superimpose all the resulting binding sites By default all aligned residues in the chains are used initially for superposition then 40 are removed for a refined superposition Optionally if y
211. nd moving the mouse in or out Similarly a translational movement of the display can be made pressing the keyboard Control button and moving the mouse with left or right mouse button depressed The same effect can be seen more easily if the Helices and Strands ribbons are replaced by their Helix Vectors and Strand Vectors For this 1fpu structure the residue numbers for the relevant DFG region are Asp381 Phe 382 and Gly383 Before inspecting this region first redisplay the Helix Vectors and Strand Vectors but with the protein Chains still off Adjust the slider at the bottom of the table until the columns for residues ASP A381 PHE A382 and GLY A383 are visible Initially toggle off the Zoom and center on clicking link tick box at the bottom left Relibase User Guide e Click the cell in the table marked PHE A382 and look at the display in the general vicinity of the ligand pyrimidine ring If there is no phenylalanine side chain visible try the active site in the other member of the dimer To do this you will need to translate and zoom the display using the keyboard Shift and Control keys e You will already have noted that under the column marking the Phe382 residue classification are a number of rows three of which have been coloured and marked with different turn types _ Helices v Helix Vectors Strands v Strand Vectors Y Ribbon Z Turns ALA A380 PHE A382 GLY A383 LEU A384 gmi __ Zoom a
212. ng one or more atoms in the template with one or more atoms in the existing substructure indicated Relibase User Guide 133 by the overlapped atoms being highlighted Once a template is in the desired position click the left mouse button to load it into the sketcher e Alternatively to load a template hit File in the top level menu of the Draw window followed by Import Template and select a template from the resulting menus e Note that protein and ligand templates contain implicit H atoms pass the mouse cursor over atoms to see how many H atoms are bonded to them Implicit H atoms are present to resolve any ambiguities that may arise e g glycine has its alpha carbon protonated otherwise glycine would match all other amino acids H atoms can be removed from All Atoms or Selected Atoms of the template if required via Atom Hydrogens Clear see Addition of Hydrogen Atoms Section 4 4 page 129 7 Substructure Display Conventions Most of the conventions and symbols used in displaying substructures are obvious Those that are not include e An atom whose symbol begins with the letter V V1 V2 etc has a variable element type Positioning the cursor over the atom will display a help message giving further details e A superscript beginning with the letter T indicates the total number of connected atoms including hydrogen atoms e g T4 indicates that the atom must be 4 coordinate e A superscript beginning with the letter X ind
213. ngless matches Coincidentally a large number of those matches that are significant contain oligopeptide like ligands which do not particularly lend themselves to the formation of drug like novel hybrids A realistic tutorial trying to demonstrate this approach requires a new cavity similarity search Relibase User Guide 4 7 ty Hermes z eal File Edit Selection Display Yiew Calculate Descriptors Help i Style Wireframe w Picking Mode Select Atoms Clear Measurements xX x y y z z 8 90 4490 9 90 9 90 2 90 2490 amp gt 4 T zoom zoom Atom selections Display Movable Chains Ligands All Entries O CavBase Query pdb1bxo 1 in reli O CavBase Hit pdb3app 1 in reli O LIM PP7_324_pdb1bxo_1 O UM PP8_324_pdb1bxq_1 O LIM XV _324 l_pdblppl_1 p rs Z rs M LIM PP4_326_pdb2web_1 O LIM 445 _3244_pdb1 ppm_1 O LIM DMF_32 7_pdb1ppk_1 CO UM ETH_14 _pdb1 ppk_1 Explore non atomic graphics objects Right click items for available options Right click Item to search for similar query cavities O CavBase Hit pdb3app 1 in reli O Cavity Surface O Active Surface C Inactive Surface O Unassigned Surface Pseudocentres Acceptor Pseudocentres Aliphatic O Pseudocentres Aromatic Pseudocentres Donor Pseudocentres DonorAcceptor Pseudocentres Pi Sec ee se se K To t
214. nt within a 3 7A radius of the water molecule in question Two descriptors for the polarity are provided water containing and water free Water containing this is calculated from the water molecule of interest to protein atoms of type O N S Cl atom metal halogen ion and aromatic carbon Water free the same as water containing however protein water molecules within 3 7A of the reference water molecule are not included in the summation e A linear cutoff function applies for specific atoms in the shell between 3 3A and 3 7A The Relibase User Guide 5 atom type weighting scheme is as follows atomtype w atomtype O 1 N 1 S 0 5 C arom 0 15 metals formal charge but always lt 2 Cl 1 always F Br I 1 if anion Coordination Geometry Ifthe water molecule has 4 or more polar atoms in its neighbourhood i e potential hydrogen bond partners the arrangement of these atoms is compared with an ideal tetrahedron normalized bond lengths are used for all calculations All permutations of 4 atom sets are superimposed onto an ideal tetrahedron and the minimum RMS deviation is reported e The average deviation from tetrahedron angles in the observed polyhedron is also reported however no descriptor value is shown if the number of neighbouring atoms is 3 or less DrugScore Energy Score The DrugScore energy score relates to the interaction energy between the water molecule and i
215. ntering the 8 Relibase User Guide crystallographic occupancy of the water molecule and the average level of B factors and occupancies in the structure see References page 172 Mobility i B fac i lt B fac gt occ i lt occ gt e Short contacts All contacts shorter than 2 4A are reported as warnings This doesn t mean there is an error in the structure it only points the user to a potential problem An almost octahedral coordination of a water molecule indicates a crystallographically misassigned atom which is more likely to be a Na or Mg atom The criteria used for notification of a potentially erroneous water molecule are The B factor of the water molecule is below 20A7 or below the average B factor in the structure There are short contacts to O and or N atoms leading to a high valence see References page 172 The RMS deviation between the best fitting polyhedron and an ideal octahedron is lt 0 25A A combination of these criteria are used to decide upon whether a water molecule is notified as being dubious The criteria for notification are displayed as shown below E Water Information for Water Molecule 13 Microsoft Internet Explorer provided by SA olx a Data Related Issues B factor 13 78 A2 Mean B factor of protein environment 21 4 A Occupancy 1 00 Mobility 0 39 Warning Short Contacts Aj Protein with contacts of 1 1 Water with contacts of 1 1 This
216. nubar In the bottom left frame there is also a Read Hitlist option see Combining and Translating Hitlists Section 6 5 page 53 To read in a plain text PDB list select PDB code from the Format pulldown click on the Browse button next to this select the appropriate file using the file browser and click Submit to load the hitlist Relibase User Guide 55 56 Relibase User Guide CHAPTER 3 RUNNING RELIBASE SEARCHES Browsing Database Entries see page 57 PDB Entry Code Searches see page 57 Keyword Searches see page 58 Protein Sequence Searches see page 65 Ligand SMILES or SMARTS Searches see page 66 2D 3D Ligand Substructure Searches see page 70 Similar Ligand Searches see page 78 Similar Protein Chain Searches see page 81 Similar Binding Site Searches and Superposition see page 84 SPMAAN SR WN 1 Browsing Database Entries e Click on the Text Search button in the Relibase menubar e Select Browse Entries from the Search Type pull down menu Now select the database i e reli or an inhouse database or hitlist to be browsed and any required resolution or X ray NMR filters to be applied e Hit the Submit button to view all database or hitlist entries that satisfy any filters set 2 PDB Entry Code Searches 2 1 Performing an Entry Code Search using the PDB e Type the required 4 character text string into the PDB Entry Code box at the top right of any Relibase page PDB entry searches are exa
217. o provided If Initial is displayed in the History column then no changes have been made to the saved hitlist If for example a hitlist has been converted from a hitlist of ligand entries to a list of PDB entry codes then HITLISTNAME L P would be given as the history e You should see the hitlists you have generated from the above searches displayed Click on the name of the hitlist e g BODE and the protein ligand entry codes are displayed in the right hand frame Selected proteins can be added to a different hitlist or removed from the current or another hitlist by selecting the appropriate buttons e Combine the 2 hitlists you have generated BODE and STUBBS by selecting BODE from 12 Relibase User Guide Protein Set 1 and STUBBS from Protein Set2 Select AND from the list of boolean operators and type in a name for the new hitlist e g COMBINED Finally hit Submit to generate the new hitlist Relibase Smiles Search Sketcher Stored Results Hitlists Relibase 2 1 0 Set Name Type Size Remove History Last Modification Content BODE Protein 159 Delete Initial Thu May 4 16 54 05 2006 ASCII XML COMBINED1 Protein 5 Delete BODE amp amp STUBBS Thu May 4 16 56 34 2006 ASCII XML STUBBS Protein 71 Delete Initial Thu May 4 16 54 49 2006 ASCII XML Combine Convert and Upload Hitlists for User robertson Protein Set 1 Operation Protein Set2 New Set Select existing hitlist z AND z Select existing hitlist z gt Sub
218. ociated with its closest pseudocentre see Pseudocentres Section 1 4 page 98 There may be small gaps between these patches i e some points on the surface may not be associated with any pseudocentre e The display of individual cavity surfaces is coupled to the display of pseudocentres as explained below e Ifthe two tick boxes Inactive Surface and Unassigned Surface in the Graphics Object Explorer see below are turned off only those parts of the surface that correspond to currently displayed pseudocentres will be displayed For instance the settings below turn on only the Donor and Donor Acceptor pseudocentres The surface can be turned off and on via the Active Surface tick box Relibase User Guide 105 aS ss Explore non atomic graphics objects Right click items for available options Right click Item to search for similar query cavities le CavBase Hit pdb1c51 3 in reli g Cavity Surface M Active Surface Inactive Surface Unassigned Surface O Pseudocentres Acceptor O Pseudocentres Aliphatic Pseudocentres Aromatic Pseudocentres Donor Pseudocentres Donor Acceptor O Pseudocentres Pi A K K Ifthe nactive Surface box is switched on those parts of the surface corresponding to undisplayed pseudocentres will be shown in green e Ifthe Unassigned Surface box is switched on those parts of the surface if any that are not asso
219. of a particular type of structure since it is often quicker to type a name than draw a substructure However substructure searching is usually better if you want to be sure of finding all examples of a particular type of ligand since ligands may be named in different ways In general and particularly in locating natural products search for only the key root part of the name e g picolin penicill This is because the names may have derivative endings Searches for trivial names drug names etc can be useful The trivial name is usually the only name stored for natural products Ligand Code Searches 3 4 1 Performing a Ligand Code Search 62 Click on the Text Search button in the Relibase menubar Select the Ligand Code option from the Search Field pull down menu Type the required text string into the Search String box Relibase User Guide Relibase PDB Entry Code I Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Text Search relibase 3 0 0 rcs Search Type Keyword z Search String NAD Match whole words only I Use regular expressions Keyword Search Options Search Field Ligand Code e g F CA 196 MCP x Filter Search Options Minimum Year Maximum Year Minimum MolVVeight Maximum MolWeight Highest Resolution A 1 0 Lowest Resolution A Structure Method Filters X ray M NMR M Use Hitlist Select existing hitlist z Save in Hitli
220. of each ligand page Similar Ligands Search launches a search of the loaded database s for ligands similar to the current ligand see Searching for Similar Ligands in the PDB Section 7 1 page 78 Similar Ligands in CSD launches a search of the Cambridge Structural Database CSD for ligands similar to the currently ligand see Searching for Similar Ligands in the CSD Section 7 2 page 80 Similar Binding Sites Search launches a search for similar binding sites see Similar Binding Site Searches and Superposition Section 9 page 84 Save Mol2 File export the ligand in mo 2 file format Save SDFile export the ligand in sd file format Save Complex PDB File export the binding site in pdb file format Save Complex Mol File export the binding site in mo 2 file format Bookmark add the current page to your list of favourites in your browser Relibase User Guide 29 4 3 Protein and Ligand Information Protein and ligand information for an acetylcholinesterase complex protein entry code lacj is shown HYDROLASE CARBOXYLIC ESTERASE QUATERNARY LIGAND BINDING TO AROMATIC RESIDUES IN THE ACTIVE SITE GORGE OF ACETYLCHOLINESTERASE Compound MOL_ID 1 MOLECULE ACETYLCHOLINESTERASE CHAIN A EC 3 1 1 7 ENGINEERED YES Reference PROC NATL ACAD SCILUSA 90 1993 9031 Author s JL SUSSMAN M HAREL LSILMAN Source MOL_ID 1 ORGANISM_SCIENTIFIC TORPEDO CALIFORNICA X RAY DIFFRACTION Cell a 113 700 b
221. oint for this type of query is always a ligand page There are eight buttons at the top of the Ligand Information page e Similar Ligands Search launches a search for ligands similar to that on the current ligand page e Similar Ligands in CSD launches a search for similar ligands in the Cambridge Structural Database to that on the current ligand page Note this search is subject to the user holding a current CSD System licence e Similar Binding Sites Search launches a search for binding sites similar to that on the current ligand page e Save Mol2 File pops up a separate browser window with a Sybyl Mol text file of the ligand e Save SDFile opens a separate browser window with an SD text file of the ligand e Save Complex PDB File pops up a separate browser window with a PDB text file of the binding site e Save Complex Mol2 File opens a separate browser window with a Mol2 text file of the binding site e Bookmark allows you to save the page so you can return to it later e In order to investigate similar binding sites click on the Similar Binding Sites Search button at the top of the Ligand Information page This will launch a form which will allow you to find all binding sites which are at least 99 identical to the one you re looking at In this example there is only one chain in close proximity to the binding site e Change the sequence identity limit in the Minimum Sequence Identity text box e If you want the liga
222. olar character but cannot participate in hydrogen bonds The definition of these includes any nitrogen that is not counted as an H bond acceptor notably planar trigonal and quaternary N and fluorine sulphur and phosphorus Rotatable Bonds A ligand bond is considered rotatable if it is single acyclic and not to a terminal atom This therefore includes e g bonds to methyl groups but not to chloro substituents It also includes bonds which although single and acyclic have highly restricted rotation e g ester linkages Finally it incorrectly include bonds to linear groups e g the bond between the methyl and cyanide carbons in CH3 CN Solvent Accessibility An atom is counted as being solvent accessible if any part of it is exposed to solvent Solvent accessibility is measured assuming a default solvent radius of 1 4A however this value can be altered Solvent Inaccessible Ligand Surface Area This is a measure of how much of the ligand surface area is desolvated upon docking calculated assuming a solvent molecule radius of 1 4A this value is the default value which can be altered The Relibase User Guide 195 surface area of the undocked ligand in the same conformation is also written out Values can be output in units of A or as percentages If percentage quantities have been requested the undocked value will always be zero Sphere Accessible Volume Two values are output before and after docking The former undocked v
223. olecule ensure its Molecule Type is set to Water see Setting Molecule Types Section 1 3 page 120 then right click on the oxygen atom of the water and pick Constrain Atom from the pulldown menu This will launch the Water Descriptor Constraints dialog box Water Descriptor Constraints B Factor 0 0 up to 1000 0 Occupancy 0 0 i hao Polarity 1000 0 up to 000 0 Contacts 0 upto i 00 Protein Contacts 0 up to 100 Ligand Contacts 0 up to 100 Water Contacts 0 up to 100 Neighbourhood Density 00 E upto hoooo OK Cancel Relibase User Guide 145 e The range for the values of crystallographic B factors polarity number of contacts and neighbourhood density can be set 12 4 Secondary Structure Constraints e The secondary structure in the protein see Secondary Structure Information Section 5 8 page 10 can be used to constrain protein substructure searches To constrain a protein atom s or residue ensure its Molecule Type is set to Protein see Setting Molecule Types Section 1 3 page 120 right click on the selected atom or area then select Secondary Structure Constraints from the pulldown menu This will launch the Secondary Structure Constraints dialog box Secondary Structure Constraints Helices Sheets amp Strands Turns Helix Properties Terminus Properties Kink Properties Helix Type 7 Ignore Helix Type Right Handed Helices L
224. ome of its dominant features highlighted in a ribbon format such as regions of helices red sheets yellow and remaining turns blue In the Assignment Method pulldown menu at the bottom of the page you will see that this display is the assignment made in the original PDB file Underneath the 3D display are a set of buttons that toggle specific features on or off Below that is a table based on individual residues for the whole of the complex that contains the information about helices and the turn type information for each of these residues In order to display this for all the complex there is a slider at the foot of the table that enables the full table to be scrolled We might start by looking to see if there is any simple qualitative difference in the assignment of helical or b sheet structure between the original PDB assessment and the SHAFT one In the interest of visual clarity use the toggle buttons to switch off the protein Chains and also the Helix Vectors and Strand Vectors The inhibitor can now be seen firmly wedged between the N terminal domain and the C block If you switch a few times between the options offered in the Assignment Method drop down menu you will note that although the b sheet assignment is the same for both methods in this instance there are some small differences in the helix assessments It may be helpful to note that the display can be zoomed by using the keyboard Shift button with the left or right mouse button a
225. omponents of proteins that are 100 homologous with the query hits numbers 54 cavity and 97 with Normalised Scores of 59 0 and 55 3 respectively You may find it interesting to look at both these cavities in Hermes superimposed on pdb1bxo 1 and speculate as to why their matches are so poor when compared to the 11 other homologous structures 4 5 Identification of sidechain backbone movement e Return to the Cavity Comparison Results table in the Relibase browser and click on the hyperlink for the matched pair containing the cavity 3app Normalised Score 55 3 Note that if you have problems locating this entry you can order the Cavity click on Cavity header in the table so that the entries are ordered alphabetically numerically 3APP is the apo form of the Relibase User Guide 5 52 1BXO complex Load the pair of cavities into Hermes as before and again toggle off the protein chains and solvent using the Protein Explorer panel Make sure that the full query surface is displayed by selecting All PCs in cavity from the Query pull down on the Display Control panel of the Cavity Control window Now separate the cavities using the slider in this same panel You will notice that although most of the back surface is matched for this pair the same cannot be said for the front You would come to the same conclusion if you slid the cavities back together again removed all the surfaces using the Graphics Objects Explorer toggled the Cavi
226. on 7 1 page 164 7 1Obtaining permissions to upload and delete structures e To register a user for permissions to upload and delete structures you will have to run the command below in a shell that has sourced the SRELIBASE_ROOT bin relibase setup sh on the server see APPENDIX E The Master Relibase Command Section page 187 S relibase dpg_user_register lt username gt lt password gt 164 Relibase User Guide To find out the names of registered workspaces run the relibase workspace_editor commands e The user to be registered must already have a workspace To check whether a user has privileges to upload and delete structures you can use the command S relibase dpg_user_check lt username gt e Note that there are also commands for removing processing priviliges from a user S relibase dpg_user_delete lt username gt and for printing a xml list of users that have data processing privileges S relibase dpg_user_print 7 2Adding a structure To add one or more structures to Relibase click on the Add Structures hyperlink on the left hand side of the data processing page and follow the instructions provided in the help box Relibase User Guide 165 Relibaset wer View Text Search Sequence Search SMILES Search Stored Results Data Processing retibase 3 1 0 Steps Add Structure s gt gt Fix Any Errors gt gt Check Templates gt gt Finished Options Add Structures m Select str
227. on atomic graphics objects This is the Graphics Object Explorer to hide surface patches unassigned The display should be similar to that below Relibase User Guide Hermes File Edit Selection Display View Calculate Descriptors Help Jighlighting W Depth Cueing 5 ShowH HideH ShowUnk Hide Unk Graphics Objects Style Wireframe Picking Mode Select Atoms B asurements x y y z z x 90 x 90 y 90 y 90 2 90 z 90 amp gt 4 T zoom zoom Atom selections a Explore non atomic graphics objects Right click items for available options Right click Item to search for similar query cavities CavBase Hit pdb2jf0 9 in reli a Cavity Surface M Active Surface Inactive Surface O Unassigned Surface Pseudocentres Acceptor Pseudocentres Aliphatic Pseudocentres Aromatic Pseudocentres Donor Pseudocentres Donor Acceptor Pseudocentres Pi CavBase Query pdb1jus 5 in reli O Cavity Surface Active Surface O Inactive Surface O Unassigned Surface Pseudocentres Acceptor Pseudocentres Aliphatic Pseudocentres Aromatic Pseudocentres Donor Pseudocentres Donor Acceptor Pseudocentres Pi E ERRARE e Check that the pseudocentre display options in the Display Controls tabbed pane of the Cavity Controls window are set to Matched PCs which they should be as this is the normal default setting
228. on both can be found on the Daylight web pages http www daylight com dayhtml doc theory index html Guidelines about the use of SMILES and SMARTS are given in the sections that follow 5 2 1 SMILES searching The following information is helpful if you use SMILES in Relibase e Information about charges isotopes and stereochemistry is ignored e Hydrogens are only allowed in brackets together with a heavy atom e g NH3 or OH e Hydrogens can be used to fill up valencies e g C O NH2 will find only carbamoyl groups and not e g peptide linkages e Relibase supports the bond type any Symbol e Relibase supports three types of atom wildcards e A Any atom This will only match hydrogen if there are hydrogen atoms stored explicitly in the ligand This is not always the case e R Any atom except H Atoms e X Any atom except C and H Atoms e Designation of aromaticity using lower case letters is supported for 5 and 6 membered aromatic rings Use single and double bonds for others e g unstaturated 5 membered rings The SMILES code can be used to designate a single aromatic bond if necessary e Relibase does not support tautomeric states Use bonds of type any SMILES code e Queries using are not supported 5 2 2 SMARTS searching The implementation of Smarts in Relibase is not comprehensive limitations are primarily due to the way in which ligands are stored in Reliba
229. opened will have a top level branch in the main tree Not all levels and branches of the tree need be displayed clicking on any icon will hide the details of the tree below that point whereas clicking on a icon will show more details The display of individual objects can be controlled by clicking on the appropriate tick boxes adjacent to those objects The surface and pseudocentre display settings made in the Cavity Controls window will automatically set the appropriate pseudocentre tick boxes in the Graphics Object Explorer Note these will be set in the individual tic boxes for each pseudocentre A tick appears in the parent tick box for a pharmacophore type if any of the underlying pseudocentres are active The tick box will be greyed unless all underlying pseudocntres are active _ ee Explore non atomic graphics objects Right click items for available options Right click Item to search for similar query cavities 2 O CavBase Hit pdb1c51 3 in reli 2 O Cavity Surface M Active Surface O Inactive Surface M Unassigned Surface Pseudocentres Acceptor Aliphatic Aromatic Donor Pseudocentres Donor Acceptor Pseudocentres Pi avBase Query pdb1fOr 2 in reli Cavity Surface Active Surface Inactive Surface Pseudocentres Pseudocentres Pseudocentres HOSNOBSAs a 0 O 1 0 SU KI Unassigned Surface a M Pseudocentres Acceptor M Pseudocentr
230. or e g X1 D2 e High precedence AND in OR subexpression e g C N amp H1 constraints can only be applied to all element types in an atom Unsupported features bond properties e Stereochemical descriptors for double bonds these are treated as single bonds with unspecified stereochemistry e High precedence AND in OR subexpression e g amp cyclic double or single and unspecified cyclicity e The following constructs are not supported e NOT any bond e g e different bond types combined with AND operator e g amp single and double e different NOT bond types combined with OR operator e g not single or not double Relibase User Guide 69 5 3 equivalent to any bond Performing a SMILES Similarity Search e Click on the Similarity Search SMILES toggle box This will activate the Minimum Similarity box Choose a minimum similarity threshold between 0 and 1 The default is 0 3 You will find that the longer the SMILES string you wish to match the higher you will need to set the threshold to avoid returning too many hits Some trial and error may be necessary The similarity threshold is a Tanimoto coefficient Tanimoto coefficients are calculated from the comparison of topological fingerprints of each ligand against that of the reference ligand The results are displayed as a list of ligands ordered in terms of similarity to the SMILES substructure The Tanimoto similarity coeffici
231. or Relibase data processing as it expects them to be marked as HOH in the PDB file If compounds such as glycerol and citrate have not been given their official three letter code this means that the ligand template matching algorithm will not be able to automatically assign them a template thus increasing the amount of manual intervention required The use of the synonyms file was designed to overcome these types of problems The synonyms file can be used to standardise the use of ligand three letter codes This is achieved by using a lookup table to allow the substitution of synonyms to a common three letter code Note that when using the synonyms file functionality Relibase will not only change the Relibase User Guide HETATM records but also the ATOM in case of synonyms of modified amino acids HETNAM HETSYN FORMUL SEQRES LINK CISPEP and MODRES records Some of these records are not explicitly used by Relibase but are modified in order to produce valid PDB files when exporting structures from Relibase No effort is made to alter three letter codes in REMARK records e The synonyms file can be found in SRELIBASE_ROOT processing synonyms txt e To make H20 D20 and TIP synonyms of HOH add the line HOH H20 D20 TIP e The first three letter code is the code that the subsequent three letter codes will be converted into 5 Customising the processing requirements e Many in
232. other type of atom Relibase User Guide 129 4 5 Note It is not possible to explicitly add hydrogens on substructures drawn with the molecule type protein see Setting Molecule Types Section 1 3 page 120 Setting Number of Connected Atoms It is possible to specify the number of connections of an atom i e the total number of atoms to which it is bonded To set the number of connections from a carbon atom In Draw or Select mode click on a carbon atom with the right hand mouse button pick Number of connections from carbon from the resulting pull down menu then select the number required from the second pull down menu Unspecified in this menu means that any number of connections is allowed When setting the number of connections from carbon all atoms will be considered including hydrogens It is only possible to specify this constraint on carbon atoms since the number of hydrogens cannot be safely inferred for other atom types To set the number of connections to non hydrogen atoms 4 6 130 In Draw or Select mode click on an atom with the right hand mouse button pick Number of connections to non hydrogen atoms from the resulting pull down menu then select the number required from the second pull down menu Unspecified in this menu means that any number of connections is allowed This constraint can be set for any atom and will consider connected heavy atoms only i e any connected hydrogen atoms will be ignored
233. ou only want to use the binding site residues from that chain make sure that the For superposition Use Binding Site Residues Only check box is selected Further options are available Click on the Submit button to superimpose the selected chains The binding site superposition is displayed in the Astex Viewer window The superposition results can also be saved in mol2 format using the Download Superimposed Structures hyperlink beneath the 3D display Textual results of the binding site superposition are tabulated Detailed information on a particular binding site can be accessed by following the relevant hyperlink in the Protein Chain section Superposition results can be saved by going to the Save Superposition Results part of the page at the bottom The results can be returned to via the Stored Results menu button Alternatively the results can be saved to a hitlist in the Save in Hitlist part of the page also at the bottom If the Automatic Visualiser Updates tick box is not enabled view the results of the superposition by clicking on the Show in Hermes button Use Hermes to have a detailed look at all components of the various superimposed complexes Hermes can be used to Relibase User Guide 1 7 e Switch parts of each binding site on and off e Colour the various parts of each binding site separately e View H bond or short range interactions There are two distinct binding modes depending on the type of ligand Find out
234. ound by the search By default the hits will be sorted in descending order of similarity score However you can click on any of the columns to sort the table on that column The table gives the following information Cavity identifier of the hit cavity Score the similarity score for the cavity from the chosen scoring function Normalised Score the similarity comparison score normalised with respect to the query cavity given as a percentage Matched centres the number of matching pseudocentres in the superimposed cavities RMS the root mean square deviation resulting from superposition of the matching pseudocentres Protein Homology the percentage sequence identity of complete protein chains defining the query and hit cavity binding site The identity is calculated for all pairs of protein chains in the query and hit cavity and the protein homology is the highest of these values Note a subtlety in the method used for the homology calculation means that only residues for which there are coordinates in the entry are included in the calculation i e the calculation may be based on fewer residues than for homology values obtained via another method either a protein sequence similarity search or from a similar binding site search This may give different homology values depending on the calculation method used Cavity Homology the percentage similarity between all the protein chains defining the query cavity and the hit cavity The
235. ox when you wish to retrieve ligands containing only the exact query SMILES string Similarity Search SMILES see Performing a SMILES Similarity Search Section 5 3 page 70 Use Hitlist allows you to use a previously saved hitlist which can be selected from the pop up menu next to Use Hitlist The default is Select existing hitlist until a hitlist is selected the entire database will be searched Save in Hitlist allows you to save the results of a search in a hitlist type the required hitlist name into the Save in Hitlist box before you start the search You will not be allowed to overwrite an old hitlist unless you click on the Overwrite Existing Hitlist button Sequence search results can also be saved after the search has been run see Creating Hitlists Section 6 2 page 48 Relibase User Guide 67 e Use Databases allows you to select which database or combination of databases is searched The databases must first have been loaded when the Relibase server was started see Section 3 of the Inhouse Data Processing manual The default setting is to search AlI databases e Hit the Submit button to start the search e The results are displayed as a list of ligands 5 2 The Use of SMILES SMARTS in Relibase SMILES are string representations of 2D molecules while SMARTS are string representations of substructures SMARTS provide variable atom bond properties and atom bond constraints which are not part of SMILES Detailed information
236. p of the form default radius is 6 0A If For Superposition Use Binding Site Residues Only is selected this choice of radius affects the residues used for the superposition In all cases it controls not only how much will be displayed in the 3D visualiser and but also what is used for superposition RMS analysis see The Analysis Table RMS Section 9 4 2 page 91 e Activate the Keep Reference Ligand Position tickbox so that the similar binding sites are superimposed on the reference ligand s original 3D coordinates if this tick box is de activated the reference ligand will be moved to the origin and all binding sites will be superimposed relative to this position e Click on the Submit button to superimpose the chains and assess protein flexibility conserved water molecules etc The results of a similar binding site search are presented in an analysis table In addition all the entries in the table are displayed in Astex Viewer in their superimposed states see Appearance of Relibase User Guide 87 Astex Viewer on a Binding Site Superposition Page Section 5 1 3 page 45 Further analysis of the superposition can be carried out using Hermes see Analysing the results in the Hermes Visualiser Section 9 3 page 88 The entries in the analysis table will either be whole protein chains or just the binding sites and ligands depending on which option has previously been selected 9 2 Saving Similar Binding Site Searches Superposition searche
237. page 125 then click on the desired spiro atom in an existing ring 126 Relibase User Guide e For example selecting a 5 membered saturated ring and clicking on one of the C atoms in en mS Pi Gisa will create 3 5 Fusing Rings by Moving One Ring onto Another e Itis possible to fuse two separate rings in the drawing area by selecting all the atoms in one ring see Selecting Atoms Section 2 7 page 123 and moving it towards the other see Moving Atoms Section 8 1 page 135 e Spiro fusion is achieved by overlapping one atom in the moveable ring with one atom in the stationary ring indicated by the overlapped atoms being highlighted Fusion will occur when the mouse button is released e Bond fusion is achieved by overlapping two bonded atoms in the moveable ring with two bonded atoms in the stationary ring It may be necessary to overlap one of the pairs and then rotate the moveable ring by holding down the Control key until the second pair overlap 4 Atom Properties 4 1 Changing the Current Element Type e The current element type determines the type of any new atom created when drawing in Draw mode The current setting is shown in the white box at the bottom of the Draw window T C H O N S P F Cl Any Other N Ligand T Single v e The current element type may be changed by hitting any of the element symbols at the bottom of Relibase User Guide 127 4 2 the Draw window Any mean
238. pdates O 367 hits for query amidin Export XML Hitlist Header BLOOD CLOTTING Save in Hitlist Title A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE Save Search Results Compound MOL_ID 1 MOLECULE THROMBIN CHAIN L FRAGMENT LIGHT CHAIN RESIDUES 328 363 SYNONYM COAGULATION FACTOR Il EC 3 4 21 5 MOL_ID 2 MOLECULE THROMBIN CHAIN H FRAGMENT HEAVY CHAIN RESIDUES 364 620 SYNONYM COAGULATION FACTOR II EC 3 4 21 5 MOL_ID 3 MOLECULE ACETYL HIRUDIN CHAIN ENGINEERED YES l e The bottom left frame displays the total number of hits found for the search To save the hitlist of ligands as an XML format file click on Export XML Hitlist In the resulting pop up window enter a name for the exported hitlist then click OK Note any saved XML hitlist can be read back into Relibase see Loading XML Format Hitlists Section 6 8 page 55 e To save the hitlist of ligands on the Relibase server click on the Save in Hitlist hyperlink In the resulting pop up window enter a name for the hitlist then click OK All ligands in a ligand hitlist can be saved in a multi mol2 or SD file via the hitlists page see Saving Hitlists Section 6 6 page 54 The search can be saved via the Save Search Results button The search can be reloaded at a later date via the Stored Results window see Storing Search Results Section 6 1 page 47 Reliba
239. ping helices and re assigning the coalesced helix to a given type via a set of hierarchical rules Fuller details of the methodology used are available see the URL above for further information 4 6 6 Viewing Secondary Structure Assignments 38 e Information about secondary structure in a protein is accessed via the Secondary Structure Information button at the bottom of protein or ligand information pages Note secondary structure shown in the main pages in Relibase is that as assigned by Astex Viewer using the DSSP algorithm rather than the assignment from the secondary structure module After clicking on the Secondary Structure Information button a Secondary Structure Information page is opened that illustrates the secondary structure assigned to the selected entry An example page is shown below for entry 1fvt Relibase User Guide Secondary Structure Information for PDB Entry 1fvt Lagands Helk Vectors Strands jn gt Y Zoot eed Center on clicking tink Tern Display All Turns X Assignment Method Orignal POB w e The page contains an instance of Astex Viewer containing the view of a given protein Beneath the Astex Viewer display are some controls for managing what is displayed Use the various tick boxes Ligands Chains Solvent Packing and Metals to switch the named component on and off Use the reset view button to return the display back to its initial view e Also provided is a
240. pseudocentres in the query cavity are deselected and appear translucent We need to choose which of them are to be included in the search query A sensible strategy is to choose all the pseudocentres in the vicinity say within 5A of the ligand To do this click with the right hand mouse button on any atom of the ligand and pick Select Pseudocentres within range of this ligand from the resulting pull down menu Type 5 0 in the resulting dialogue box and hit OK e The 3D display now shows the portion of the cavity that the search will try to match The picked pseudocentres are depicted solid 34 Relibase User Guide 3 4 Hit the Search button at the bottom right of the Cavity Controls window to complete the query definition This should open a browser page that will enable us to set some other search options Selecting Search Options and Running the Search We do not want to search the whole database so click on the down arrow icon to the left of the Select an existing hitlist box and select from the resulting pull down menu the hitlist that we created earlier which you may have called TUTORIAL Type in a search name e g tutorial Ijus Delete the 00 0 from the Maximum permitted homology between proteins box and type in 20 0 instead By doing this we ensure that any hits we find will have low sequence homology with QacR i e we would not have been able to find them by more standard sequence based methods The point of this exer
241. r Guide in the sketcher window e The current molecule type may be changed by clicking on the button at the bottom of the Draw window and selecting from the resulting pull down menu C H O N S P EF Cl Any Other Cc Ligand ba Single v e Itis also possible to allow substructures to be either Protein or Ligand Protein or Water or Ligand or Water molecule types To allow a substructure to be either Protein or Ligand or Water select the molecule type Any Substructures of these mixed types are drawn in grey e The selected molecule type will determine the type of any new atom created when drawing in Draw mode There are a number of ways to change the molecule type of an existing substructure including e In Select mode Select the substructure that you wish to change see Selecting Atoms Section 2 7 page 123 then change the current molecule type using the pull down menu at the bottom of the Draw window In any mode right click on an atom or bond within the substructure and from the resulting menu select either Place Fragment in Ligand or Place Fragment in Protein e To set the molecule type of all atoms in any mode pick Atoms from the top level menu and select either Place All Atoms in Ligand or Place All Atoms in Protein from the resulting pull down menu Relibase User Guide 121 2 Fundamentals of Drawing 2 1 2 2 122 Drawing a Bond Ensure you are in Draw mode see Modes in the Drawing Window
242. r the carbon to nitrogen bonds and ensure that the molecule type is set to Ligand e Change the molecule type to Protein and draw the carboxylate group with Any bond type for the carbon to oxygen bonds e Set up distance constraints between the N and O atoms by clicking on the Add 3d button to the left of the drawing area Select the atoms involved in the constraint i e N and O hit Define next to Distance in the Add 3d pop up window Once you have defined the first pair of atoms do similarly for the remaining N and O atom Relibase PDB Entry Code Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Heip 2D 3D Sketcher relibase 3 1 0 File Edit View Atom Bond Options Help Query Sketcher 3d Parameters Click to place atom click and drag to draw bond D1 D2 Add 3d X1 PR r View Controls ks X1 af START SEARCH C HO N S P F Ct Any Other c Protein w Bond Single v e Now define the angle between the planes of the two fragments To do this first select the N C N atoms and hit the Define next button next to Plane in the Edit 3D Parameters window In the same way define the plane for the O C O moiety To define the angle between these planes select Plane1 and Plane2 in the Edit 3D Parameters window then hit Define next to Angle e Click on Done to close the window Relibase User Guide 25 26 Hit Search to start the search In th
243. rch Results Selecting one of these cavities will then give you further details of that cavity and load it into the visualiser see Displaying and Comparing Cavities Section 3 page 101 Relibase User Guide 4 6 Secondary Structure Information e The term secondary structure describes the general 3D form of local segments of the protein i e amino acids Secondary structure in proteins is typically mediated by H bonding interactions between amino acids e The methodology involved in the display of secondary structure in Relibase is covered in the sections that follow 4 6 1 Introduction e Secondary structure is assigned to protein structures to derive a sense of the relative fold of one protein with respect to another There are many examples of assignment protocols published in the literature The most widely used method is known as Define Secondary Structure of Proteins or DSSP Kabsch amp Sanders but others also exist e Secondary structure assignments usually operate by recognising particular intra molecular non bonded features between given residues in a protein Pairs of residues that exhibit these pre defined features are then assigned as being a component of a helix or sheet Strands and turns are also observed and can be sub components of helices or sheets In raw PDB entries information is presented about helices and sheets Some turn information is also presented but the data are on average usually limited to a
244. rch results from the following types of searches can be stored as hitlists Text searches see Keyword Searches Section 3 page 58 Results from this type of search can be selected to be saved either before or after the search has been run Sequence searches see Protein Sequence Searches Section 4 page 65 Results from this type of search can only be selected to be saved before the search has been run In addition to being initiated from the Relibase menubar this same search can be started from the Similar Protein Search box in the Protein page vide infra e SMILES SMARTS searches see Ligand SMILES or SMARTS Searches Section 5 page 66 Results from these types of searches can be selected to be saved before or after the search has been run 48 Relibase User Guide 2D 3D searches see 2D 3D Ligand Substructure Searches Section 6 page 70 Results from this type of search can be selected to be saved before or after the search has been run e Similar ligand searches see Similar Ligand Searches Section 7 page 78 Results from this type of search can only be selected to be saved after the search has been run e Similar protein chain searches see Similar Protein Chain Searches Section 8 page 81 Results from this type of search can only be selected to be saved before the search has been run e Binding site superpositions see Similar Binding Site Searches and Superposition Section 9 page 84 Results from this type of search ca
245. re but turns that are similar in nature tend to have assignment names that are similar For example below is the set mean torsion angles for d turns reverse 3 residue turns types Ia and Ib are relatively similar the major difference is in y 2 all other torsion angles have overlapping ranges cluster type j r y W j A Y gt f f f f f 1 Ta 124 0 21 0 51 0 273 175 9 6 4 126 7 34 2 155 1 15 5 2 Ib 116 2 17 7 49 8 18 2 174 4 7 4 148 6 22 7 169 5 12 6 3 Ha 127 4 21 6 26 3 22 2 176 4 6 8 87 0 29 4 148 5 24 9 4 IIb 104 8 10 0 24 5 25 6 174 1 4 6 105 2 8 7 172 8 3 6 5 Ha 113 2 196 274 30 1 177 5 5 3 124 2 32 7 162 9 15 1 6 IIb 131 8 21 6 42 2 35 5 175 0 6 2 84 5 30 8 160 8 15 8 7 Hic 166 2 11 1 32 7 23 9 176 5 0 1 127 5 30 0 163 9 0 2 8 IVa 148 4 13 8 98 2 12 4 0 4 3 9 74 0 12 6 159 7 15 3 9 IVb 131 7 23 4 115 5 79 9 7 13 3 57 9 9 173 1 5 1 e In cases where a given turn sub classification resultant from ESOM clustering corresponds to a previously defined sub classification the name used corresponds to that in the literature So for example g turns normal 3 residue turns are classified into inverse or normal sub classifications e A full description of all turn types is available in the publications provided in the Classification Methodology see page 35 4 6 5 SHAFT Assignment of Helices e Once turns have been assigned it is relatively straight forward to build up a given sequence of tu
246. rent criteria 1st Sort Backbone orSidechein 2nd Sort Pseudocentre Type 3rd Sort l Residue Type Column 1 PseudoCentres Backbone Acceptor M Donor Pi Sidechain Metal M Acceptor Aliphatic Aromatic Donor a ARG GLN Donor Acceptor he Search 1 K K 8 8 8 p e T The tick boxes may then be used to select particular types of pseudocentres e g the following settings indicate that donor pseudocentres in Asn and Asp residues are selected Cavity Controls Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1f0r 2 in reli Hit CavBase Hit pdb101m 1 in reli er Display Controls Search Setup Use any of the methods below to select pseudocentres PCs in the Query cavity to be searched for Jattclicking on PCs rightclick on objects to pick nearby PCs clicking on the tick boxes below Use the pulldown options below to sortthe pseudocentres by different criteria 1st Sort Residue Type _ g 2nd Sort Pseudocentre Type i 3rd Sort Backbone or Sidechain Column 1 PseudoCentres o O ALA Acceptor Aliphatic O Donor Pi ARG Aliphatic Donor M Pi ASP M Acceptor Aliphatic v Search E K E E E K a
247. residue turns ASP L14 GLN L144 THR L14B GLU L14C LYS L14D GLU L14E e Beneath the table of secondary structure assignments there are a number of other options e Zoom and center on clicking link keep this tick box checked if you wish to highlight centre and zoom the selected secondary structure element uncheck the tick box if you wish to only highlighted the secondary structure element the state of the centring and zoom will be unaffected Relibase User Guide e Turn Display select options in this pull down menu to control whether to display All Turns Rare Turns all turns except those that are parts of a helices or No Turns e Assignment Method use this pull down menu allows to alternate between helices as pre assigned in the original PDB file compared to those assigned using the SHAFT methodology Turn information is always presented using results from the SecBase assignment e 2D 3D searches can be constrained so that the resultant hits contain specific secondary structure elements see Defining Secondary Structure Elements Section 13 page 146 4 6 7 Helix assignments from the PDB versus SHAFT assignment notable differences Using the SHAFT for building helices for the most part results in assignments that are broadly similar to PDB assignments The major differences so far noted are in the termini of helices Further SHAFT assigns more a helices e Helical Termini e Generally SHAFT ten
248. rietary structural data This document describes the process of uploading in house structures into Relibase by translating PDB files into Relibase database entries e The Relibase data processing system has been designed to be highly flexible and easy to use The main reason for this is that there are many different scenarios for uploading protein structures into Relibase A protein crystallographer uploading a single structure has got different requirements to a molecular modeller uploading a backlog of 3000 structures Both these scenarios are catered for in the Relibase data processing system 2 Overall workflow e Conceptually data processing can be broken down into two steps 1 Ensure the PDB file is in an acceptable format required as in house structures may deviate from the PDB file format 2 Ensure the ligand atom and bond typing are acceptable required because the PDB file format does not contain information on ligand bond types e Several features have been implemented to make the above tasks as painless as possible e Data processing converts PDB files into Relibase database entries The first step of the data processing parses the PDB file and detects any errors or missing fields These are reported back to the user who is prompted to fix them After any errors have been corrected the user will have to confirm any ligands that do not already have templates Ligand templates are required to ensure correct atom and bond typing
249. rmation Section 4 6 page 35 Customisable content and hyperlinks to external resources can also be added see the Relibaset Installation Notes Appendix B Attp www ccdc cam ac uk support documentation relibase Relibase User Guide Relibaset eget view Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Ligand Information for R56_1 L pdb1bhx reivase 3 0 0 server re2 Similar Ligands Search Similar Ligands in CSD Similar Binding Sites Search jl Save Mol2 File lI Save SDFile lI Save Complex PDB File Save Complex Mol2 File Bookmark Ligand R56_1 L PDB entry 1bhx Chemical name 5 OX0 6 PHENYLMETHANESULFONYLAMINO HEXAHYDRO THIAZOL O 3 2 A PYRIDINE 3 CARBOXYLIC ACID 3 GUANIDINO PROPYL AMIDE y Ligands Chains v Solvent Y Schematic Show Embedded Visualiser M Width 500 Height 350 Apply Hermes Controller Showin Hermes Automatic Visualiser Updates E Header SERINE PROTEASE Title X RAY STRUCTURE OF THE COMPLEX OF HUMAN ALPHA THROMBIN WITH THE INHIBITOR SDZ 229 357 MOL_ID 1 MOLECULE ALPHA THROMBIN CHAIN A EC 3 4 21 5 MOL_ID 2 MOLECULE ALPHA THROMBIN CHAIN B EC 3 4 21 5 MOL_ID 3 MOLECULE ALPHA THROMBIN CHAIN F EC 3 4 21 5 MOL_ID 4 MOLECULE ALPHA THROMBIN CHAIN E EC 3 4 21 5 Compound Additionally the following buttons are present at the top
250. rmation before this entry can be added to Relibaset Please review the files in SRELIBASE_ROOT processing dp_cmd PRE_TEMPL Edit if required then copy to SRELIBASE_ROOT processing TEMPL Then re process your input file e At this stage you will have to manually validate the templates in SRELIBASE_ROOT processing dp_cmd PRE_TEMPL before copying them to RELIBASE_ROOT processing TEMPL To re process your input file re run the the first command given above i e relibase data_process input path to dir 2BYH pdb database aaa mtz path to dir 2byh_sigma mtz template path to dir 2BYH_2D7 mol2 8 2Processing multiple PDB files in a directory e It is possible to provide a directory with PDB files to process However we would discourage the use of this option as it is more cumbersome Suppose that you had some structures in home olsson structures and you wanted to add them to the database aaa The command below would be used to start the process Relibase User Guide 169 relibase data_process input home olsson structures database aaa e Note that at this stage it is highly likely that some of your structures will not have been added to the database because Relibase wants you to check your ligand templates These are located in the SRELIBASE_ROOT processing dp_cmd PRE_TEMPL directory unless validation is swi
251. rns into a larger secondary structure element e The general feature of a helices is the intra helical NH CO 4 hydrogen bonding so that each residue within a helix is involved in two backbone hydrogen bonding NH CO 4 and CO NH 4 The first and last four residues within an a helix are only involved in one type of backbone hydrogen bonding leaving four NH groups at the N terminus and four CO groups at the C terminus that can interact with other partners e g other parts of the protein or water Relibase User Guide 37 Therefore in an ideal a helix the N3 position would be the last residue with this free backbone NH at the N terminus and the C3 position would be the first residue with a free backbone CO group at the C terminus The assignment of the N capping and C capping position using turn motifs is thus based on this free backbone groups and the analysis of specific turn types at the terminus This approach was used to convert sequences of turns into helical elements If one recognises the turn types that occur in helices then one can build up sequences of contiguous turns that are of the appropriate type and assign them to a specified helix type Helix capping residues can be assigned as those that are at the ends of the contiguous sequences Some issues with overlapping helices of different types were identified where for example an a helix would be overlapped with a 3 9 helix This was resolved by coalescing the overlap
252. rom the water and the cavity modules of Relibase Hermes can be accessed from all protein and ligand pages by clicking the Show in Hermes button However some users simply want a quick way of loading the protein structures into their favourite Relibase User Guide 179 visualiser For this purpose there is a Save PDB File button at the top of the protein pages and a Save Complex PDB File button at the top of ligand pages The behaviour of these download buttons can be customised in your web browser to automatically open the downloaded files in a 3rd party visualiser of preference Opening pdb2e1a_full_entry pdb You have chosen to open 3 pdb2g1a_full_entry pdb which is a PDB file From http devO1 ccde cam ac uk 8080 What should Firefox do with this File open vah Save File M Do this automatically For files like this from now on Settings can be changed using the Applications tab in Firefox s Options e Finally note that on top of protein pages that have structure factors associated with them there is a button named Save PDB MTZ File This can be used to download a tar file containing both the PDB file and the associated MTZ file By writing a custom script to handle this archive it is possible to set up a system where PDB files are automatically displayed with their associated electron density maps in a visualiser such as Coot by pointing the browser at a script such as the 180 one outlined below bi
253. rs Highest resolution A Lowest resolution A 125 0 v Search X Ray Structures Z Search NMR Structures Allow intra chain contacts v Search DNA Structures Superposition file generation Batch search Run batch search with name Start Cancel e Highest Resolution and Lowest Resolution boxes can be used to filter on the experimental precision of X Ray DNA or NMR derived structures that you wish to consider If you only wish to consider X Ray structures with a resolution of 2 0A or better then enter 2 0 into the Highest Resolution box If only Search X Ray Structures is toggled on then a Lowest Resolution can also be set All NMR structures by default have a resolution set of 1 0 and cannot be filtered in this way Note when restricting a search to DNA structures either the Search X Ray Structures or Search NMR Structures tick box must be activated e Structure Method Filters can be used to restrict the search to either X Ray DNA or NMR derived structures The default is that no restriction is made Toggle off the criterion that is not required e If you have created a search which contains two fragments either ligand ligand or ligand protein then the Search packing environment check box in the contact filters area will become active If this box is checked then it means that the search will also include the situations where one of the fragments is part of a neighbouring protein packed c
254. ry and make it a drug like structure bearing in mind the sorts of parameters that would influence bioavailability or metabolic stability is of course a little more problematic If you look at the superimposed ligands from 2oah 2web 2vj9 and lepr you may get some idea of how such a set might help produce ideas for a non peptide penicillopepsin inhibitor Pairwise comparison between cavity protein homologies and no of PC matches It is sometimes interesting to compare these matched homologies with each other and with their corresponding scores and no of PC matches For example it could be useful to identify receptors with little overall sequence identity to the known query but which are highly homologous in those residues around the cavity and perhaps therefore functionally equivalent although this sort of information is likely to be already known using other methodologies More realistically it would be useful to identify structures with little direct cavity homology but a significant number of PC matches structures that might increase the chance of finding matched cavities that do not have already known equivalent functionality where bound ligand overlays may provide novel insights towards de novo design Relibase User Guide 55 e Given that the data set searched in this tutorial was preselected on the basis of known functionality aspartic proteases and acid proteinases this search you have just conducted is unlikely to provide a good e
255. s rightclick on objects to pick nearby PCs clicking on the tick boxes below Use the pulldown options below to sort the pseudocentres by 2 x different criteria 1st Sort Backbone orSidechain v 2nd Sort Pseudocentre Type F 3rd Sort Residue Type x Column 1 PseudoCentres 5 M Backbone H A Acceptor Donor aM Pi e At the foot of the Cavity Controls window press the Search button e A Cavity Search Setup webpage will be launched where cavity search parameters can be configured We will discuss later some of the factors that may be considered when considering the number and type or spatial density of preferred PCs for any specific query However for the purposes of this tutorial we will search using 36 pseudocentres e From the Select an existing hitlist pulldown menu select the tutorial hitlist you saved earlier e Modify the Number of solutions to save top N value of 100 to 400 e Change the Select a Scoring Function radio button to scoring function 3 e Keep all other settings at their defaults The Cavity Search Setup window should resemble the following Relibase User Guide 47 Relibaset PDB Entry Code f view Text Search Sequence Search SMILES Search Stored Results Cavity Search Setup relibase 3 0 0 Rc5 Similarity Search for Cavity pdb1bxo 1 using 36 Pseudocentres Parameter Setting Select an existing hitlist
256. s Properties Kink Properties Helix Type v Ignore Helix Type Right Handed Helices JAI E O All Left Handed Helices All O An Other Helix Length Minimum Length oH Maximum Length 1 000 4 Reset Reset Constraints 1 Helices Sheets amp Strands Turns m Ok Cancel e Using this dialog it is possible to constrain the secondary structure of the amino acid to be in either a sheet a helix or a turn e The dialog is separated into 3 major sections e Helices see Defining Helix Properties Section 13 3 page 148 e Sheets amp Strands see Defining Sheet and Strand Properties Section 13 4 page 150 e Turns see Defining Turn Properties Section 13 5 page 152 Relibase User Guide 147 e Use the Reset button in each of the separate sub section above to clear the pane that is currently on view and return it to its original settings e Use the Reset Constraints buttons at the bottom of the dialog to reset all constraints of a given class to the default 13 3Defining Helix Properties e Within the Helix Properties pane it is possible to constrain on the basis of the properties of a particular helix type and length The Helices tab is subdivided into three panes e Helix Properties e Terminus Properties e Kink Properties e By default original PDB assignments of helices are when one specifies a helix constraint This can be changed to use the SHAFT
257. s any atom i e an atom of any element type and is denoted by the symbol Any in the drawn substructure Other displays a pull down menu Choosing Select from periodic table allows selection of any element in the Periodic Table Right click on the canvas ensuring no atoms are selected click on Set Element Type and follow the pull down menu to the appropriate atom type Setting Variable Element Types Atoms in a substructure may be variable e g F or Cl or Br or I Any means any atom i e an atom of any element type and is denoted by the symbol Any in the drawn substructure The current element type see Changing the Current Element Type Section 4 1 page 127 may be made variable by hitting the Other button at the bottom of the Draw window This displays a menu from which common variable element types e g Any Metal Halogen etc can be selected Alternatively choosing Select from periodic table from this menu opens up the Periodic Table Ce Pr Nd Pm Sm Eu Gd Th Dy Ho Er tm vo jo Th Pa U Np Pu Am Cm Bk Cf Es Variable Type History Reset Apply OK Cancel Java Applet Window e From here it is possible to create your own variable element type by clicking on the required 128 elements e g O and S Pre defined element groups can also be used by selecting the appropriate group symbols from the periodic table Relibase User Guide e Hit Apply to ac
258. s can be saved as hitlists or the search results stored To store the search results e Go to the bottom of the results page and enter a name for the stored search in the Save Superposition Results area Add a description of the superposition if necessary and then click on Save You will not be allowed to overwrite existing saved superposition results unless you click on the appropriate toggle box e Superposition results can be retrieved via the Stored Results button on the menu bar at the top of the Relibase page Stored superpositions may also be deleted on this page e To save a hitlist e Go to the bottom of the results page and enter a name for the hitlist in the Save Hitlist area then hit Save You will not be allowed to overwrite an existing hitlist unless you click on the appropriate toggle box 9 3 Analysing the results in the Hermes Visualiser 88 e For information on using Hermes please refer to the Hermes documentation follow the Hermes link on the top right of this document Relibase User Guide E Relibase Protein Visualiser File Selection Displ ji Iculate Help Highlighting idrogens Style Wireframe w Picking Mode Select Atoms x x y y z z x 90 x 90 y 90 y 90 2 90 2 90 amp S J T zoom zoom Protein Explorer Display Movable gt Metals Al Entries 140 1 E SUSI jgands
259. se 5 Ligand SMILES or SMARTS Searches 5 1 Performing a SMILES SMARTS Substructure Search e Click on the SMILES Search button in the Relibase menubar e Type the required Smiles or Smarts string of the substructure you wish to search into the Enter SMILES SMARTS Code box 66 Relibase User Guide Enter SMILES SMARTS Code e g NC Njctecece Minimum MolvVeight Maximum Mol Veight Highest Resolution A 0o Lowest Resolution A o Structure Method Filters X ray M NMR M Exact Match SMILES I Similarity Search SMILES Minimum Similarity p 3 Use Hitlist Select existing hitlist _v Save in Hitlist Overwrite Existing Hitlist Use Databases Submit e Various Options are available Minimum MolWeight and Maximum MolWeight boxes can be used to restrict the molecular weight and size of ligands that you wish to retrieve from your search Leaving these boxes empty means that all ligands are considered Highest Resolution and Lowest Resolution boxes can be used to filter on the experimental precision of X Ray derived structures that you wish to consider If you set a highest resolution of anything other than 1 0 or empty no NMR derived structures will be retrieved Structure Method Filters can be used to restrict the search to either X Ray or NMR derived structures The default is that no restriction is made Toggle off the criterion that is not required Exact Match SMILES activate this check b
260. se The following should be taken into consideration when using Smarts e Relibase assumes bond types given in the SMARTS query match Relibase conventions In 68 Relibase User Guide particular e Six membered aromatic rings have aromatic bond types however complete 6 membered rings in SMARTS input with single double bond types will be converted to aromatic e Five membered rings are non aromatic unless pi bonded to a metal e g ferrocenes e Due to the nature of the data source hydrogen counts on atoms other than carbon are not reliable use of Dn atom constraint number of non hydrogen connections is recommended rather than Xn total number of connections for heteroatoms Unsupported features general e Dot disconnected fragments e g C C e Recursive SMARTS e g CC CCC e Reaction SMARTS e g CC gt gt CC Unsupported features atom properties e Some atom constraints where n is an integer e v lt n gt valency constraint e x lt n gt number of ring connections constraint e h lt n gt implicit hydrogen constraint no distinction is made between implicit and explicit H in Relibase e Charge constraints no charges are stored in Relibase e R lt n gt where n gt 1 no smallest set of smallest rings implementation e n atomic number the element symbol should be used e lt n gt atomic mass e Stereochemical descriptors e Constraints of different types combined with OR operat
261. se User Guide 23 3 4 Viewing Sequence Based Search Results 24 The results of sequence based searches are displayed as a table of chains Relibaset PDB Entry Code f view Text Search Sequence Search SMILES Search Stored Results Similar Chain Sea rch Results Relibase 3 0 0 server rc2 Hits 1 40 of 508 Chain Alignment Ligands 100 7 aa pdb2pks C pdbi1bhx F pdbtriw C Na Na pdb2hnt F No Ligands e The first column lists the Relibase protein entry code together with a chain identifier e g entry pdb2pks chain C Click on the entry code to go to the Relibase protein entry page see Relibase Protein Entry Pages Section 4 1 page 27 for this structure The second column lists the percentage identity with respect to the reference chain and the length of the matched sequence Above in pdb2pks a sequence match of seven amino acids 7 AA has been made with the query sequence For sequence searches see Protein Sequence Searches Section 4 page 65 the reference chain is that part of the sequence typed into the sequence search box Click on the percentage identity to analyse the alignment For a typical sequence search the results look like this Relibase User Guide Relibaset PDB Entry Code view Text Search Sequence Search SMILES Search Stored Results Seq uence Alignment Relibase 3 0 0 server rc2 Alignment of Entries pdb2pks C and Query Seque
262. se consists of only one polypeptide chain To find all proteins with an amino acid chain that is identical to the one you are looking at i e the Torpedo Californica acetylcholinesterase 10 Scroll down the 1E3Q protein information page to the Protein Chains part The different protein chain sequences can be displayed by clicking on the appropriate chain hyperlink e g pdb1a3q A Select the Submit button next to Search for Similar chains Tip searches for similar or identical protein chains can be invoked from any protein page The Minimum Sequence Identity and Maximum Sequence Identity boxes can be used to specify the required sequence identity as a percentage with respect to the reference chain default is 100 If you wish to display the ligand 2D chemical diagrams in the resulting list of chains select the Show Ligands check box The results are displayed as a table of chains ranked according to their sequence identity relative to the reference chain Relibase User Guide 1 5 Relibaset Heip Keyword Search Results relibases 2 2 0 rc3 pabiac Z pdbtaci Hats 1 40 of 56 pdbiamn l pdbiaxs gt Pil Chain Alignment Ligands 0 f oon Ao TRK pdbitsu 100 0 pobidxe A 47 aa pdbikus odbimaa z 88 hits for query acetylcholinesterase port XML Hitlist The links from the sequence identity in this table will show the alignment of two complete chains Conserved residues
263. ser inspection however indicates an absence of strong H bond acceptors in the receptor around the end of this extension that might stabilise such fragments Indeed most ligand fragments that one might envisage filling this part of the cavity would have to be quite long and flexible and consequently not strongly bound For the purpose of this tutorial we will remove the PseudoCentre PC points associated with this region of the receptor In fact given that the pentapeptidomimetic ligand 1s itself quite large when compared with many pharmaceutically interesting ligands we shall reduce the full set of 90 pseudocentres identified in the cavity to just those within 4 0 Angstrom of the existing ligand in the query cavity e Click the Search Setup tab in the Cavity Controls window Relibase User Guide 45 46 Cavity Controls 2 x You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1bxo 1 in reli Hit None Display Controls Search Setup Use any of the methods below to select pseudocentres PCs inthe Query cavity to be searched for Jettalicking on PCs nightclick an objects te pick nearby PCs clicking on the tick boxes below Use the pulldown options below to sort the pseudocentres by different criteria 1st Sort Backbone orSidechain gt 2nd Sort Pseudocentre Type gt 3rd Sort ResidueType gt PseudoCentres Backbone
264. sidues this sequence identity applies to and in cases where a match is impossible without the inclusion of insertions in the matched sequence the number of insertions that are required Chains with maximum identity number of homologous amino acids and fewest insertions are ranked highest e The search results can be stored by typing a name and description of the search into the relevant boxes in the Save Similar Sequence Results section of the results window at the bottom of the page then hitting Save The stored search can be viewed at a later date via the Stored Results window see Creating Hitlists Section 6 2 page 48 Note the Fasta sequence search finds the 1000 best sequence matches in the sequence files then filters the resulting chains on homology When searching for low homologies the required chains may not be within the 1000 best sequence matches found by Fasta 4 2 Hints for Sequence Searching e The searches are not case sensitive e The searches are done via the FastA program Only those hits are returned that are considered significant by FastA If given long strings FastA will also return hits for which only a subset of the original search string is matched For a detailed description of FastA the user is referred to the FastA user manual see http fasta bioch virginia edu e Information on installing FastA is provided in the Relibase installation notes http www ccdc cam ac uk support documentation reliba
265. sition To move the complete query i e the entire contents of the sketcher window use the rotate buttons in the View Controls section to the left of the Draw window View Controls ala Ara Z _ ja m im Re Relibase User Guide 8 4 Duplicating Substructures Copy and Paste e To make a copy of all or part of a substructure select see Selecting Atoms Section 2 7 page 123 the atoms and bonds to be copied e Click on a blank point in the white area with the right hand mouse button and select Copy or hit Edit in the top level menu and Copy in the resulting pull down menu Alternatively use the keyboard shortcut CTRL C A copy of the selected substructure or part substructure will appear in the sketcher The substructure copy should be moved into the desired position using the mouse To scale the substructure move the mouse while keeping the Shift button depressed to rotate move the mouse while keeping the Control button depressed To fuse the substructure copy with an existing substructure of the same molecule type move it towards the substructure Fusion is achieved by overlapping one or more atoms in the substructure with one or more atoms in the existing query indicated by the overlapped atoms being highlighted Once a copied substructure is in the desired position click the left mouse button to load it into the sketcher 9 Reading Saving and Deleting Queries e To save a query set up in the sketcher select File
266. st NAD Overwrite Existing Hitlist Submit e One two and three letter ligand entry codes can be searched using this method e g Ca will return all PDB entries with calcium metal ions present and F will return all PDB entries with fluorine counter ions Note that all characters in the search string will be matched i e this is not a substring match AC will not match ACE but regular expressions can be used e g AC e Various additional Options are available for all text based searches However not all options are available for all searches see Options for Text Based Searches Section 3 5 page 64 e Hit the Submit button to start the search The results are presented as a browsable list of ligands The search text will be highlighted in red within the Chemical name of each ligand The ligand and corresponding binding site are displayed in the AstexViewer window The search results can be saved to a hitlist after the search has been run using the Save in Hitlist button in the bottom left hand frame of the results window 3 4 2 Hints for Ligand Entry Code Searching The entry codes are assigned to so called HET groups in the structure A ligand can be built up of more than one HET group each with its own entry code One common situation is where the ligand is a polypeptide chain in which case each amino acid in the chain is a HET group and is Relibase User Guide 63 3 5 64 represented by the standard 3 letter co
267. st bit is derived from a hash code that accounts for elemental type of the current node and the and the path already traversed The second bit is derived from a hash code that only accounts for atom types traversed along the current path The Tanimoto coefficient is set to a default value of 0 7 During a similar ligand search only ligands with a Tanimoto coefficient relative to the reference ligand above this threshold value will be displayed e The 2D diagrams in the second column are linked to the corresponding ligand pages e The search results can be filtered on the basis of the Tanimoto coefficient Enter the required minimum similarity index a value between 0 and 1 the default value is 0 7 into the Minimum Similarity window and hit the Submit button e Output options are available Export XML Hitlist use this button to save the hitlist of ligand entry codes as an XML format file Note any saved XML hitlist can be read back into Relibase see Loading XML Format Hitlists Section 6 8 page 55 Relibase User Guide 79 7 2 e Save in Hitlist use this button to save a hitlist of ligand entry codes onto the Relibase server Searching for Similar Ligands in the CSD On any ligand page click on the Similar Ligands in CSD button on the menu bar above the 2D ligand diagram All ligands in the CSD are compared to the reference ligand The results are loaded into WebCSD the online interface to the CSD http www ccdc cam
268. st need to create our small subset hitlist using a keyword search So hit the Text Search button select Keyword from the Search Type drop down list and type acid proteinase into the Search String box Because we wish to include some structures that are NOT necessarily acid proteinases ensure the Use regular expressions tick box 1s activated Note activating the Use regular expressions tick box means you will retrieve structures that have comments like the following string in the header column metalloproteinase with the amino acid sequence Without changing any other settings enter a suitable name in the Save in Hitlist dialogue box e g aspartic protease then hit the Submit button to run the search and save the results to a hitlist Repeat the process but this time enter aspartic protease into the Search String box and give the resultant hitlist a different name We now need to combine these two hitlists Hit the top level Hitlists button In the Hitlist Operations frame select your first hitlist from the dropdown menu under Protein Set 1 Similarly select your second hitlist from the dropdown under Protein Set 2 and then combine the two by selecting the OR command from the Operation dropdown menu Enter a suitable name e g tutorial into the New Set box and hit the Submit button to create the new combined hitlist When the job is done there will be a tutorial hitlist stored which we will use as the database subset for our cavity sear
269. stick format e It might perhaps be of interest to repeat this process in a different browser window with a conventional ATP binding site inhibitor complex for example the structure of the tyrosine kinase ACK1 1u54 and compare the similarity and differences e A quick initial inspection of the ligand filled active site of 1fpu suggests that the DFG out protein is in part stabilised by good overlap with a neighbouring aromatic ring in the inhibitor It might therefore help to find similar DFG conformations if we add a simple additional distance Relibase User Guide 63 5 6 64 requirement to our search and look for a ligand with an aromatic ring close to that of the Phe382 However a closer look at the complex could lead to many other conclusions about additional 3D constraints that would probably be characteristic of this allosteric site for example a lipophilic ligand portion in the region vacated by the Phe382 ring would probably be as good an identifier as the one we have selected And lastly because the DFG out pocket fillers are all kinase inhibitors and we want this search to be quick we will limit the search to known kinase structures e Prepare the kinase subset by clicking on the Relibase menu option Text Search e Select Keyword from the Search Type pulldown menu and type kinase into the Search String box e Type key_kinase into the Save in Hitlist box select reli in the Use Databases box then hit the Submit button
270. sults used to access results from previous searches binding site superpositions see Similar Binding Site Searches and Superposition Section 9 page 84 similar sequence searches see Protein Sequence Searches Section 4 page 65 and cavity similarity searches see Cavity Similarity Searching Section 4 page 107 e Help provides access to the Relibase User Guide and technical documentation The Relibase Home Page When you first access the Relibase server the Relibase Home Page will be displayed The Home Page can also be accessed from any Relibase page by hitting the Home button in the top menubar Relibase User Guide 17 18 Relibaset PDB Entry Code Home Text Search Sequence Search SMILES Search Stored Results Relibase Homepage Relibase 3 0 0 server rc2 Relibase A program for searching protein ligand databases Install 3D Visualization Software Client Workspace Administration In house Database Building Tool Cavity Similarity Results Camments bugs queries to Cambridge Crystaliographie Data Centre 12 Union Road Cambridge C82 7EZ S United Kingdom gt tel 44 7223 336022 WWW Dito www cede cam ac uk e mail support eccde cam ac uk Current workspace henderson Databases currently loaded gregtest reli ssetest i For by Diagram generation in Relibase is powered by ChemAxon s Marvin Software EB chemAxon m astex Embedded visualisation in Relibase is powered by
271. t to catch ligands that have already had their atom and bond types assigned which is particularly useful for common crystallisation reagents such as glycerol and citrate If the three letter code does match a template in the re i database the template and the ligand are compared using substructure matching This step filters out any false positives where a ligand in the PDB file has for some reason or other been given a three letter code matching a different compound in reli If a ligand does not match an entry from the re i database the ligand is matched against all user supplied templates provided during the data input Matching against the user supplied templates is also performed using substructure matching If no hit is found processing performs an advanced automatic determination of what the atom and bond typing should be This auto typing can be automatically accepted by setting the auto_accept_template flag to true in the relibase_processing conf file Alternatively if the auto_accept_ligand_template flag is set to false the user will be prompted to manually validate the ligand template before it is accepted Relibase User Guide 159 Note that user supplied templates should be in mol2 file format 4 Synonyms file 160 A problem that can occur with in house PDB files is that compounds such as water citrate and glycerol are given differing three letter codes over the years In terms of water molecules this presents a problem f
272. t to the N cap residue Similarly the C one C two residues follow a similar pattern e Dectivate the Ignore N Terminus Properties or Ignore C Terminus Properties check box to enable and define N Terminus and C Terminus properties e Use the Reset button to return the settings to their original display e Note the capping residues for helices are Neap to Niwo and Ctwo to Ceap for 349 helices Neap to Nihree and Cthree tO Ceap for a helices Neap tO Nihree and Cihree tO Ceap for p helices Kink Properties Relibase User Guide 149 Helices Sheets amp Strands Turns Helix Properties Terminus Properties Kink Properties v Ignore kink type One Kink Two Adjacent Kinks Reset e Use the Kink Properties tab to define and search for amino acids in kinks e Kinks are points in helices where the direction helical vector mapping along the centre of the helix from the N cap to the C cap change The secondary structure module allows such kinks to be searched for either where one change of direction occurs or where two adjacent changes of direction occur e Deactivate the Ignore kink type check box to enable and select kink properties to be searched for e Use the Reset button to return the settings to their original display e Note the kink properties are assigned to the midpoint of the Ca atoms of four adjacent residues You can define one of these Ca atoms in the case of one kink
273. t you want to process a large number of in house structures In this case the preferred method is to create a wrapper script around the single entry command line tool This is particularly effective if you have mol files of the ligands associated with the structures that you want to upload In these cases a script along the lines of the following Python script would be ideal e If you do have a large number of in house structures to process and are unsure of how to go about this please do not hesitate to contact support ccdc cam ac uk 8 4Using an alternative configuration file e In cases where people have different types of quality or source of PDB files it might make sense to specify a set of configuration files For example suppose that a molecular modeller wanted to create an in house database consisting of PDB files derived from a molecular dynamics MD 170 Relibase User Guide simulation The MD derived PDB file might be lacking most PDB header records and as such would need a more permissive set of configuration file settings In this example the modeller might set the auto_accept_template_ligand flag to t rue and disable the requirement of all the missing records in the MD PDB file Suppose these settings were saved in a file called path to md_processing conf and the MD snapshot PDB files were located in a directory called path to structures the modeller could then upload all the files to a database called md_snapshots using the comma
274. tabase Section 4 4 1 page 112 Monitoring Search Progress Aborting Searches see Monitoring Search Progress Aborting Searches Section 4 5 page 112 Browsing the Results of a Search see Browsing the Results of a Search Section 4 6 page 113 4 4 1 Searching a Subset of the Database 4 5 112 Cavity similarity searches can take a very long time so if you know in advance that you only want hits from certain types of proteins e g kinases you should confine the search to a subset of just those entries To set up a subset you need to create a hitlist Subsets or hitlists that can be searched are protein or ligand hitlists or hitlists that have been converted to cavity hitlists Note it is no longer essential to convert a protein or ligand hitlist to a cavity hitlist prior to using the hitlist as a cavity search subset Start by performing a Relibase search to find the entries you want e g a text search for the keyword kinase Save the results in a protein hitlist if you are searching on a protein property or a ligand hitlist if you are searching on a ligand property Select the relevant protein or ligand hitlist from the Select an existing hitlist pull down menu It will then be used as a subset for the cavity similarity search Monitoring Search Progress Aborting Searches Once you have started a cavity similarity search you will see a display something like this Relibase User Guide Progress of Similarity Searching
275. tched off e After you have validated your ligand templates you need to move them from the SRELIBASE_ROOT processing dp_cmd PRE_TEMPL directory to the S RELIBASE_ROOT processing TEMPL directory You can then add your structures to the database using the same command as used previously Fy H relibase data_process input home olsson structures database aaa Note that if you forget to move or copy the templates from the RELIBASE_ROOT processing dp_cmd PRE_TEMPL directory to the RELIBASE_ROOT processing TEMPL directory before you re run the command no additional structures will be added to the database as the templates need to be in the latter directory to be regarded as accepted e If you want to get a set of structures from a particular directory into a Relibase in house database and you do not want to validate the ligand templates this can be achieved by setting the auto_accept_ligand_template flag to true in the relibase_processing conf file e To re iterate we discourage the use of this option as it is more cumbersome than the single entry command line option The single entry command line option can be used to upload a large backlog of in house structures see Processing a large backlog of in house structures Section 8 3 page 170 8 3Processing a large backlog of in house structures e Suppose tha
276. the Drawing Window Section 1 2 page 120 Ensure the molecule type is set to that of the existing atom i e protein ligand or water see Setting Molecule Types Section 1 3 page 120 Move the cursor onto the atom Press down the left hand mouse button Move the cursor while keeping the mouse button depressed then release the button Drawing a Bond to an Existing Atom Ensure you are in Draw mode see Modes in the Drawing Window Section 1 2 page 120 Ensure the molecule type is set to that of the existing atom i e protein ligand or water see Setting Molecule Types Section 1 3 page 120 Relibase User Guide 2 5 2 6 2 7 Move the cursor into the white area of the drawing window Press down the left hand mouse button Move the cursor onto the desired atom the bond locks onto the atom while keeping the mouse button depressed then release the button Drawing a Bond between Two Existing Atoms Ensure you are in Draw mode see Modes in the Drawing Window Section 1 2 page 120 Ensure the molecule type is set to that of the existing atoms i e protein ligand or water see Setting Molecule Types Section 1 3 page 120 It is not possible to add bonds between different molecule types Move the cursor onto the first atom Press down the left hand mouse button Move the cursor onto the second atom the bond locks onto the atom while keeping the button depressed then release the button Undoing Mistakes when Dr
277. the hit protein sorted in descending order by the score of those cavity matches compared to date The results page is updated every 15 seconds After 15 20 minutes the search will have completed The top of the Search Results page should look like this Relibase User Guide Relibase PDB Entry Code SI Home Text Search Sequence Search SMILES Search Sketcher Hitlists Stored Results Help Cavity Comparison Search Result retibase 3 0 0 rcs Comparison for Query Cavity pdb1bxo 1 Label search_pdb1bxo 1_36_1 Search settings used maximum permitted homology between proteins minimum permitted ligand size in cavity minimum score to save number of solutions to save scoring function search all entries in the hitlist tutorial NAM henderson Search Results 1 400 of 400 2 Normalised Matched Protein Cavity Select Cavity Score Score Centres RMS Homology Homology Header Title r db1bxa 1 5806 0 375 35 100 0 100 0 HYDROLASE ACID PROTEINASE PENICILLOPEPSIN COMPLEX WITH PHOSPHONATE INHIBITOR CRYSTALLOGRAPHIC ANALYSIS OF TRANSITION STATE MIMICS m pdblppl1 5141 7 86 4 HYDROLASE ACID PROTEINASE BOUND TO PENICILLOPEPSIN PHOSPHORUS CONTAINING PEPTIDE ANALOGUES ACID PROTEINASE PENICILLOPEPSIN E C 3 4 23 20 COMPLEX WITH PHOSPHONATE INHIBITOR METHYL 2S 1 N FORMYL L o db2web 1 50547 84 3 eee VALYLJAMINO 2 2 NAPHTHYLJETHYLJH
278. thods Specify whether hit cavities are to be rejected if they are not occupied by ligands of at least N atoms Type in the minimum permitted score hit cavities will only be kept if their similarity with the query exceeds this value Unfortunately the different scoring functions are on different scales so you will need to get some experience before you can use this option effectively Specify the maximum number of hits that you want e g the top 50 Specify the maximum allowed resolution Specify the Search priority value 10 highest 19 lowest priority This option effectively allows you to determine how important searches are so that they either run quickly top priority or run in the background without interfering with other tasks low priority Select a scoring function see Similarity Searching and Scoring Section 1 5 page 99 At present the performance of these functions has not been fully characterised so you may need to experiment to find which one works best for any particular query All of them seem reasonably reliable at CCDC we usually use scoring function 3 Relibase User Guide 111l Hit Start Search to begin the search Searches may take many hours it is safe to close down your Relibase session while a search is running i e it will not stop the search and you can collect the results later from a new session Related topics Searching on a Subset of the Database Cavity Hitlists see Searching a Subset of the Da
279. tion 2 page 57 e Text searches see Keyword Searches Section 3 page 58 e Author name searches see Author Name Searches Section 3 2 page 59 e Ligand compound name searches see Ligand Compound Name Searches Section 3 3 page 61 Ligand entry code searches see Ligand Code Searches Section 3 4 page 62 e Database browsing capabilities see Browsing Database Entries Section 1 page 57 e Sequence Search provides access to Searches on amino acid sequences see Protein Sequence Searches Section 4 page 65 e SMILES Search provides access to e Searches on ligand SMILES or SMARTS strings see Ligand SMILES or SMARTS Searches Section 5 page 66 Sketcher provides access to e 2D 3D ligand substructure searching see 2D 3D Ligand Substructure Searches Section 6 page 70 e Non bonded protein ligand or protein protein interaction searching see Non bonded interaction searching Section 6 6 page 72 Some Relibase searches can only be started from Relibase protein entry pages see Relibase Protein Entry Pages Section 4 1 page 27 or from Relibase ligand pages see Relibase Ligand Pages Section 4 2 page 28 these include Ligand similarity searching see Similar Ligand Searches Section 7 page 78 e Similar chain searching see Similar Protein Chain Searches Section 8 page 81 e Similar binding site searching and superposition see Similar Binding Site Searches and Superposition Section 9 page 84 Relibase Us
280. tion page clicking on this button takes you to a page displaying the volume of the cavity containing the ligand header information and the ligand chemical diagram if available Also Hermes will open with the selected cavity loaded see Displaying and Comparing Cavities Section 3 page 101 e If you are on a Protein Information page clicking on the Cavity Information button takes you to a page listing all the cavities in the protein structure with their volumes and any ligands they contain clicking on a ligand diagram links you to the Ligand Information page Selecting one of these cavities will then give you fuller details of that cavity and load it into Hermes Cavities in PDB Entry 1a4g db1a4g 1 pdb1a4g 2 pdb1a4g 3 pdb1a4g 5 Similarity Search Results e Ifyou have created a cavity hitlist see Searching a Subset of the Database Section 4 4 1 page 112 and want to view one of the cavities in it display the hitlist manager page by clicking on the top level Hitlist button and then click on the name of relevant cavity hitlist e g esterase _cav below This will display all the entries in the hitlist 100 Relibase User Guide Relibaset PDB Entry Code i view Text Search Sequence Search SMILES Search Stored Results Hitlists Relibase 3 0 0 server rc2 Set Name Owner Type Size Access Remove Last Modification Content Hits 1 40 of 3506 berglig toby_iw_deb4 Ligand 6 Public Not owner Mon Aug 11 2008 1
281. tions H Gohlke M Hendlich G Klebe J Mol Biol 295 337 356 2000 Cavity Information Module CavBase LIGSITE Automatic and efficient detection of potential small molecule binding sites in proteins M Hendlich F Rippmann and G Barnickel J Mol Graph Model 15 359 63 389 1997 From Structure to Function A New Approach to Detect Functional Similarity among Proteins Independent from Sequence and Fold Homology S Schmitt M Hendlich and G Klebe Angew Chem Int Ed Engl 40 3141 3144 2001 A New Method to Detect Related Function Among Proteins Independent of Sequence and Fold Homology S Schmitt D Kuhn and G Klebe J Mol Biol 323 387 406 2002 Structural Aspects of Binding Site Similarity A 3D Upgrade for Chemogenomics In Chemogenomics in Drug Discovery Eds Hugo Kubinyi and Gerhard M ller Wiley VCH Weinheim 2004 A Bergner and J G nther Secondary Structure Module SecBase Turns revisited A uniform and comprehensive classification of normal open and reverse turn families minimizing unassigned random chain portions O Koch and G Klebe Proteins Structure Function and Bioinformatics 74 353 367 2008 DOI 10 1002 prot 22185 Secbase Database Module To Retrieve Secondary Structure Elements with Ligand Binding Motifs O Koch J Cole P Block G Klebe J Chem Inf Model 49 2388 2402 2009 Prediction of turn types in protein structure by machine learning classifiers M Meissner
282. tlist Submit e Various additional Options are available for all text based searches However not all options are available for all searches see Options for Text Based Searches Section 3 5 page 64 Hit the Submit button to start the search e The results are presented as a browsable list of Relibase entries The author s name that makes up the query will be highlighted in red in the Author s field The search results can be saved to a hitlist after the search has been run using the Save in Hitlist button in the bottom left hand frame of the results window 3 2 2 Hints for Author Searching The searches are not case sensitive e Searches for Huber will also hit Glockshuber unless the Match whole words only box is ticked 60 Relibase User Guide e You should be aware that the searches are based entirely on the bibliographic information given in the original PDB file e If you wish to include authors initials you should use M Harel i e with no space between the initial s and the surname e Itis possible to search for two authors s names simultaneously Multiple author names should be separated by a space 3 3 Ligand Compound Name Searches 3 3 1 Performing a Ligand Name Search e Click on the Text Search button in the Relibase menubar e Select the Keyword option in the Search Type box e Select the Ligand Compound Name option from the Search Field pull down menu e Type the required text string into th
283. to start the search A hitlist called key_kinase will be saved Building the 3D search query Launch the sketcher by clicking on the top level Sketcher button When the sketcher appears build the query until the page looks as below Note that we have assumed familiarity with the sketcher and the construction and definition of 3D constraints such as centroids and distance ranges Also the atomic distinction between protein and ligand atoms Please refer to earlier tutorials or to the Relibase documentation for further information Relibase User Guide Relibaset PDB Entry Code PA Home SMILES Search Hitlsts Stored Results Help 2D 3D Sketcher Relibase 3 0 0 RC5 File Edit View Atom Bond Options Help Modes j Query Sketcher 3d Parameters Click on atom to select click and drag to select several atoms AC maa Search a Templates Other View Controls ay 3 lt ul gt ARA i Options iT gl Delete N a a C H 6 N Ss P F CI Any Other C Ligand single M e Within the Edit 3D Parameters window launched when the Add 3D button is clicked define the ring centroids then define the distance between them constraining them to a lower limit of 1 0A and to an upper limit of 6 0A e Make sure that the atoms of the DFG fragment have been typed as protein blue and those of the other aromatic ring as ligand type blac
284. ton underneath this list e In the resulting pop up type the new name into the Label input box e g 3D Parameter Options x Label D1 Lower Limit 2 0 Upper Limit 3 5 OK Cancel Relibase User Guide 143 e Alternatively you can rename a parameter immediately after creating it by hitting the Options button in the Geometric Parameters dialogue box opened by hitting Add 3d 11 5 Displaying Geometric Parameter in the Draw Window e A defined parameter may be displayed by clicking on its name in the 3d Parameters list top right hand corner of Draw window 11 6 Deleting a Geometric Parameter e A parameter may be deleted by clicking on its name in the 3d Parameters list top right hand corner of Draw window and then clicking on the Delete button underneath this list 12 Applying Constraints 12 1 Geometric Constraints 144 3D substructure searches are performed by defining relevant geometric parameters see Defining Geometric Parameters Involving Atoms Section 11 2 page 140 and constraining their values To constrain the value of a defined parameter If you have just defined the parameter so that the Geometric Parameters dialogue box 1s already open hit the Options button If the Geometric Parameters dialogue box is not already open select the parameter you wish to constrain in the 3d Parameters list top right hand corner of the Draw window and hit the Options button underneath t
285. toolbox and looking at the available Packing check boxes This can be of importance when looking at protein ligand complexes and assessing the binding sites in structure based drug design If crystal packing is present then you need to assess if the ligand conformation is the physiologically relevant one and is not distorted as a result of crystal packing effects This also has an application in protein ligand docking when using e g GOLD and the incorrect ligand conformation is predicted when crystal packing effects are not taken into consideration Including crystal packing allows GOLD to predict the correct ligand conformation Since the reference structure 1 fax does not contain any water molecules it cannot be used to carry out the analysis for conserved water positions By using the Change Reference Chain button you can select a structure containing water molecules thus enabling the analysis to be Relibase User Guide performed Relibase User Guide 19 20 Relibase User Guide 2 Tutorial 2 Substructure Searching in Relibase SMILES and 2D 3D 2 1 Introduction A key feature of Relibase are substructure based 2D and 3D searches Searches for ligand substructures can be carried out using the SMILES search form More complex searches in addition to simple searches can be set up in the Relibase 2D 3D query sketcher 2 2 2D Ligand Substructure Searching SMILES or 2D 3D 2 2 1 Search for Ligands Comprising Different Su
286. tory USA who maintained and developed the PDB archive until 1998 e The members of the Research Collaboratory for Structural Bioinformatics RCSB responsible for the maintenance of the PDB http www rcsb org pdb since 1998 Relibase User Guide 175 176 Dr Thomas Mietzner BASF Ludwigshafen Germany for providing code for the superposition of molecules based on atom pairs The German Federal Ministry of Education and Research BMBF Merck Darmstadt Germany BASF Ludwigshafen Germany and Boehringer Ingelhheim Germany for funding the RELIWE and RELIMO projects Dr Stefan Schmitt in the group of Gerhard Klebe at the Institute of Pharmaceutical Chemistry Philipps University Marburg Germany for the cavity information module CavBase Dr Oliver Koch jointly at CCDC and in the group of Gerhard Klebe at the Institute of Pharmaceutical Chemistry Philipps University Marburg Germany for the secondary structure information module SecBase Relibase User Guide APPENDIX A Keyboard Shortcuts for the Relibase Sketcher e Keyboard shortcut options that are available for the Relibase sketcher are provided below Keyboard Shortcut CTRL A CTRL I CTRL SHIFT A Delete CTRL C Relibase User Guide Function Selects everything in the sketcher see Selecting Atoms Section 2 7 page 123 Everything that is selected in the sketcher becomes unselected and vice versa see Selecting Atoms Section 2 7 page 123
287. ts local environment The energy score is calculated from knowledge based potentials which have been derived from the observed preferences for particular atom pair interactions e By implementing a new atom type for water oxygen atoms special potentials describing preferred types of water protein and water ligand interactions have been derived analogously to DrugScore see References page 172 All contact pairs up to a length of 6 0A in a selected dataset were used to derive the potentials e The total score is calculated from the individual protein water ligand water and water water contributions All contributions are displayed in the form of coloured bars as shown below and the units are unscaled DrugScore units 6 Relibase User Guide Water Information for Water Molecule 70 Microsoft Internet Explorer mE x Descriptors for Water Molecule 70 atom 2433 in Entry pdb1a3e Visualise Binding to Local Environment Number of Polar Contacts Protein Ligand Water Local Polarity and Geometry Polarity of environment 3 400 Polarity of environment water free 2 752 Energy Scores Protein Ligand Water Total eas 2658 8 6411 601 5559 606 14630 008 5 6 2 Local Topology of the Protein Structure The protein topology is described in terms of e Neighbourhood Density see page 8 e SAS Solvent Accessible Surface see page 8 Relibase User Guide 3 Water Information for Water Molecule 70
288. ture then you can upload the Mal2 ligand template file s Process Files Manfred Hendlich 1994 1999 and Cambridge Ciystaliographic Data Centre 1999 2011 WS wac Si 7 4Deleting a structure or a database e To delete a structure or a database click on the Delete hyperlink on the left hand side of the data processing page and follow the instructions provided in the help box Note that to delete a single entry you will need to provide the full name of that entry the full name is taken from the name of the PDB file uploaded to Relibase Relibase User Guide 167 Relibaset PDB Entry Code SN Text Search SMILES Search Stored Results Data Processing relibase 3 1 0 Options Database Entries Add Structures Single Structure All Delete O 5 SS User test Single Entry code Log Out A Delete an entire user Delete database or a single entry Manfred Hendlich 1994 1999 and Cambridge Crystallographic Data Centre 1999 2011 WEES WC panh e Using the settings above all entries in the database inhouse _db would be deleted when the Delete button is clicked on If only a single entry was required to be deleted e g inhouse_structurel pdb then the Single radio button should be activated indicating only one entry is being deleted The filename of the entry you wish to delete should then be entered into the Entry code box i e inhous
289. tutorial X search_pdbibxo 1_36_1 Maximum permitted homology between proteins hoo Soo Minimum ligand size N atoms oc Minimum score to save oo o o Number of solutions to save top N Search name Maximum resolution permitted Search priority nice value 10 highest 19 lowest priority scoring function 1 scoring function 2 scoring function 3 Select a Scoring Function Start Search Press the Start Search button 4 3 1 Some Observations on Dealing with the Search Results 48 After starting the cavity similarity search a new Relibase page will appear the Cavity Search Status page which after about one minute changes again to highlight the current status of the search and a second table that restates the search settings used Three options are available at the foot of the page e Click here to view all search results view previous search results whose data have been saved e Click here to view current results for this query view current search results e Finish this query now stop the search Note that search results obtained to this point will be saved and can be viewed After a few further minutes click the second option to view the current results for this query A Search Results page will appear with data on Score Matched Centres number of matched pseudocentres RMS RMS distance between matched pseudocentre pairs Protein Homology and Cavity Homology and header details about
290. ty Controls display to show only Matched PCs then rotate the ligand Well matched gt back wall e LA Lack of equivalent matches in front Display the protein chains for both cavities H atoms undisplayed and remove the ligand from the query protein Gently rotating the display will make it immediately obvious that some of the amino acid residues superimpose quite closely on top of their counterpoints whilst a few do not Select one atom for each of those residues on the 1bxo query receptor that doesn t superpose well and then label each of those residues You should see labels on Gly76 Asp77 and Gln111 and to a lesser extent on Tyr75 and Ser79 All of these are on the front wall although you may also note a single bond rotation has taken place on Glu16 on the back Note these numbers and select each complete residue using the top level menubar pull down Selection Define Complex Selection By residue Specify Individual and then clicking on those residues 1bxo and not 3app before adding them to the selection by selecting Add and then Close then Close again to finish the selection The corresponding atoms will now be highlighted in the 3D view Colour them distinctively right click in open space in the 3D view select Colours and then e g Orange perhaps changing style to Capped Sticks Inspection of the result indicates quite clearly that the inside of the receptor cavity has collapsed quite signi
291. ty searching e Similar chain searching e Similar binding site searching and superposition e WaterBase access e CavBase access Relibase Text Based Searches 1 4 1 Performing an Entry Code Search Click on the Text Search button in the Relibase menubar Type lacj into the Search String box PDB entry searches are exact match searches which will match on the given string only i e a search on pdb et will not retrieve pdb etr Entry Searches Search Type PDB Entry Code x Search String 1acil Submit Hit the Submit button under to the Search String box to start the search The results are presented as a single Protein Information page the protein will be displayed in the Astex Viewer at the top of the page The protein structure can be analysed in more detail by launching Hermes To do this click on the Show in Hermes button in the Hermes Controller part of the Protein Information page Reliview Controller Show in Reliview Automatic Visualiser Updates I Whether or not the viewer is updated when navigating through Relibase can be controlled using the Automatic Visualiser Updates check box A summary of the textual bibliographic and crystallographic information for the entry is given in the body of the page Relibase User Guide 5 HYDROLASE CARBOXYLIC ESTERASE Ti Unavailable Compound ACETYLCHOLINESTERASE C 3 1 1 7 COMPLEXED WITH TACRINE itle PROC NAT ACAD SCLUSA 90 1993 9031 J LSUSSMAN M
292. ubmit e Hitlists may be subtracted from any loaded database e Select the database you wish to subtract the hitlist from the Database 1 pulldown list e Minus will already be selected in the Operation pulldown menu e Select the hitlist you wish to subtract from the Set 2 pulldown menu e Type the name of the new hitlist and press Submit A new hitlist of the same type i e Protein Ligand or Cavity will be added to the list 6 6 Saving Hitlists e Entries in ligand hitlists can be saved by viewing the hitlist then selecting one of the following options Hits 1 40 of 1611 gt Bi Ligand Set nad_lig View Entries Save Ligand Multi Mol2 File Save Complex Multi Mol2 File Save Ligand SDFile Save Ligand Spreadsheet Add to Set nad_lig2 Submit Remove from Set nad_lig Submit Make New Set Submit I NAP pdb2j8z T NDP pdb1u72 T NDP pdb 1xkq I NAP pdb2fr8 e View Entries this button re loads the search results into the browser window e Save Ligand Multi Mol2 File use this button to save all hit ligands to a multi mol file e Save Complex Multi Mol2 File use this button to save the ligand and its binding site to a multi mol 2 file 54 Relibase User Guide 6 7 6 8 6 9 e Save Ligand SDFile use this button to save all ligands to an SDFile e Save Ligand Spreadsheet use this button to save ligand information for all hit ligands to a
293. uctures for processing Single Structure Delete Files Browse User test Log Out m Database New Select the files that you wish to add to Relibase using the Browse button Process Files You can either add the structures to a new or an existing user database To start processing of the new structures press Process Files Manfred Hendlich 1994 1999 and Cambridge Crystallographic Data Centre 7999 2077 WS RF vac agp 7 3Adding a structure with associated MTZ and or ligand templates e To add a PDB file with associated MTZ and or ligand templates click on the Single Structure hyperlink on the left hand side of the data processing page and follow the instructions provided in the help box 166 Relibase User Guide Relibase PDB Entry Code a Data Processing relibase 3 1 0 Steps Add Structure s gt gt Fix Any Errors gt gt Check Templates gt gt Finished Options Add Structures m Select structure for processing Single Structure Delete File Browse User test Log Out m Reflection data optional MTZ File Browse This form allows you to attach additional information to a single structure m Ligand template files optional Files Browse To store electron densities specify an MTZ file m Database If you have already determined the atom and New Tr bond typing for any ligand in the struc
294. ueries see page 137 10 Geometric Objects see page 138 11 Geometric Parameters see page 139 12 Applying Constraints see page 144 SPMAAN SR WN 1 Sketcher Basics 1 1 Layout of the 2D 3D Drawing Window Open the substructure drawing window by clicking on the Sketcher button in the Relibase menubar File Edit View Atom Bond Options Help Modes Query Sketcher i 3d Parameters f Click to place atom click and drag to draw bond Templates O O 2 Other oA N CH ONSP F C Any Other c Ligand vy Single v Relibase User Guide 119 10 11 1 2 Top level menu Mode buttons responses to mouse clicks in the drawing area will depend on which mode is active see Modes in the Drawing Window Section 1 2 page 120 Buttons to set up geometrical parameters and constraints see Geometric Parameters Section 11 page 139 Button for starting searches see Running a Search Section 6 8 page 74 Menu for selecting templates molecular building blocks to aid drawing see Using Substruc ture Templates Section 6 page 133 View controls buttons to translate rotate and re size the display Drawing area see Sketcher Basics Section 1 page 119 Area for changing the current element type see Changing the Current Element Type Section 4 1 page 127 Menu for selecting molecule type water ligand or protein see Setting Molecule Types Section 1 3 page 120 Area for listing displaying
295. ulate Descriptors Help Highlighting v Dep eing Stereo ShowH HideH ShowUnk Hide Unk Graphics Objects Style Wireframe Picking Mode Select Atoms Clear Measurements x x y y z z x 90 x 90 y 90 y 90 z 90 z 90 amp gt J T zoom zoom Atom selections Launch this on receiving a cavity You can also show this dialog using the menu available by right clicking on items in the Graphics Object Explorer Query CavBase Query pdb1f0r 2 in reli Hit None Display Controls Search Setup Show Pseudocentres Hit e Some instructions on how to manipulate cavities and initiate cavity searches are given in the following sections For more information on viewing modifying and manipulating structures and other items in the Hermes visualiser please refer to the relevant section of the manual follow the Hermes link on the top right of this document 3 2 Moving Objects in the 3D Display 3 2 1 Rotating Translating and Scaling e The 3D display can be rotated by moving the cursor in the display area while keeping the left hand mouse button pressed down x and y rotation or while keeping both the left hand mouse button and the Shift key pressed down z rotation e If you have a mouse with three buttons or two buttons and a scroll wheel the contents of the display area can be translated by moving the cursor while holding the middle mouse button or scroll wheel down Alternatively use the left
296. uld now be displayed in the protein environment of the query cavity The display should look something like this Relibase User Guide The ligand ortho 7 that resides in the hit cavity in acetylcholine esterase has an orientation in the query cavity that looks credible the ligand has been made more prominant by displaying it in capped stick mode To investigate the steric fit more closely click with the right hand mouse button on the CavBase Hit select Styles and then Spacefill Whether or not this ligand would actually bind to QAcR is open to question of course but we are alerted to the possibility The tutorial can be finished at this point but if you are interested the next section will demonstrate a few more of the viewer options 3 6 Experimenting with Other Visualiser Options The Graphics Object Explorer provides control of the display and the colour of pseudocentres and surfaces First activate the active surface for the hit cavity by clicking the corresponding Active Surface tick box in the Graphics Object Explorer Click with the right hand mouse button on items in the Graphics Object Explorer select Colours and select a colour from the pull down menu to change the colour of a surface this is not possible for an Active Surface or a pseudocentre Relibase User Guide 4 e In assessing the complementarity of the query cavity surface and the ligand it can help to focus in on just one particular part of the sur
297. umber of PDB entries number of ligand templates and number of ligand models for the currently loaded databases can be found by following the Database Statistics link from the Help menu A list of all PDB entries which have been excluded from Relibase with associated reasons are available in SRELIBASE ROOT etc Refused_PDB entries list 5 Database Information content The Relibase database contains all the information stored in the original PDB files Searchable information fields are described in the following sections 5 1 5 2 5 3 5 4 Bibliographic Information Authors names Publication date Deposition date Not searchable Textual Information The PDB HEADER COMPND and SOURCE records Experimental method X ray or NMR Sequence Information Amino acid sequence of protein chains Chemical Information Ligand compound name Ligand entry code 2D chemical connectivity used for 2D and 3D substructure searches non bonded interaction searches Ligand Molecular weight Crystallographic Information Unit cell parameters Not searchable Space group Not searchable Relibase User Guide e Resolution e Crystal packing of the protein ligand binding site 5 6 Water Molecule Descriptors Relibase includes a series of descriptors which are precalculated for each individual water molecule and stored in a database The descriptors are described in the following sections 5 6 1 Binding of a Water to its Local En
298. using a proxy server Relibase will additionally need to be configured with your proxy server settings in order to communicate with the EDS server To do so edit the JAVA_OPTS line in your SRELIBASE_ROOT bin relibase setup config file You Relibase User Guide 183 will need to ensure that the following settings are present Dhttp proxyHost my proxy com Dhttp proxyPort 1234 where my proxy com and 1234 are your actual proxy server and port number For example JAVA_OPTS Xmx512m Xms256m verbose ge Dhttp proxyHost my proxy com Dhttp proxyPort 1234 e Note that to make any changes to the relibase setup config file take effect you will need to stop your relibase server source your Relibase setup and restart the relibase server Electron Density Viewing e Viewing the electron density maps can give an immediate appreciation of the quality of the experimental data upon which a structure is based e Electron density maps can be displayed in the web based visualiser by clicking on the Load Maps button By default both the 2FoFc blue and the Fo Fc ve green ve red maps are displayed If you want to hide any of the maps deselect the relevant checkboxes in the controller e The result of a successful X ray crystal structure determination is the 3 dimensional distribution or density map of the electrons in the crystal However as the electrons are closely associated with the atomic nuclei the electron dens
299. vironment The binding of water to its local environment can be described in terms of e Number of Polar Contacts see page 4 e Polarity of the Local Environment see page 5 e Coordination Geometry see page 6 e DrugScore Energy Score see page 6 Number of Polar Contacts e This set of descriptors reports the number of polar atoms i e the potential hydrogen bond partners within a 3 3A radius of the water molecule in question The total number is split up into polar contacts to protein ligand and other water molecules respectively e Atoms taken into account are O N and Cl atoms halide anions metal cations For O and N atoms contact distances shorter than 2 4A are considered but will also result in a warning not for metal cations e The number of protein ligand and water contacts are displayed using a colour coded bar 4 Relibase User Guide E Water Information for Water Molecule 70 Microsoft Internet Explorer mE x Descriptors for Water Molecule 70 atom 2433 in Entry pdb1a3e Visualise Binding to Local Environment Number of Polar Contacts Protein Ligand Water Local Polarity and Geometry Polarity of environment 3 400 Polarity of environment water free 2 752 Energy Scores Protein Ligand Water Total ed 2658 8 6411 601 9559 606 14630 008 Polarity of the Local Environment e This is distance dependent and is a scaled measure of the polarity of the local environme
300. what characterises these two binding modes Look at the positions of the water molecules to help find these Investigate the results shown in the analysis of superimposed chains summary table Investigate how flexible the main and side chains are Are there any conserved water molecules in the binding site Consider changing the reference by selecting the Change Reference Chain button and select the Recalculate Table button to create a new table Investigating Crystal Packing Effects superposition of binding sites can be carried out including crystal packing effects thus allowing investigation of the influence of crystal packing on the ligand binding mode within a series of related structures The aim of this example is to search for crystal packing effects in factor Xa binding sites and find out which structures are influenced by crystal packing effects Click on the Text Search button in the Relibase menubar Type fax into the Search String box ensuring the Search Type menu is set to PDB Entry Code Search then hit the Submit button Go to the Ligand Information page for DX9 in the retrieved PDB entry for 1 fax and click on the Similar Binding Sites Search button as in the previous example In the resulting page accept the default values and start the search for similar chains by clicking on the Submit button In the options which are available for chain selection click on With Ligands Only which will exclude entries with no ligan
301. would have let you know that neither neighbouring protein chains nor additional ligands are involved so the Contact filters do not need to be activated We do however wish to search only a kinase subset of the Relibase database and therefore need to select the tab marked Hitlist Controls Select the key_kinase hitlist that you prepared earlier from the pull down menu marked Restrict search to hitlist named Enter an appropriate name for the search e g secst ructtutorial in the Save search in hitlist named window and select reli from the list of databases provided in the Search in database domains window In this particular search you haven t specified any atoms onto which the search hits are to be superimposed and because this search is fairly fast 15 25 mins there is no need to run it as a batch job so the options below do not have to be switched on Relibase User Guide e Click on Start to start the search e When the search is finished a new Search Results page will open e Use the Save Search Results option at the bottom left of this page to save the results of this search e The resulting hitlist is a list of Tyr Ser Thr kinase allosteric inhibitors It will not be exhaustive though A cursory inspection of the matches found might suggest a query that includes an essential lipophilic contact with the Cbatom of the DFG Asp and a good H bond NH donor contact with an a helical SHAFT classified glutamic acid residue found on the mo
302. ww phenix online org both with and without fill column names 2FOFCWT 2FOFCWT_no fill PH2FOFCWT PH2FOFCWT_no fill FOFCWT PHFOFCWT e If you have MTZ files that use different column names please get in contact so that we can add the capability to parse them e There are no dependencies for uploading and storing MTZ files However in order to view electron densities in the web based visualiser CCP4 http www ccp4 ac uk software and libraries are required see APPENDIX C Electron Density Configuration and Viewing Section page 183 Relibase User Guide 163 7 Processing structures using the web based GUI e To upload or delete structures from your in house database s click on the In house Database Building Tool hyperlink on the Relibase home page This will prompt you for a username and password Relibaset PDB Entry Code Text Search Sequence Search SMILES Search Stored Results Data Processing relibase 3 1 0 Options Please log in to access data processing Add Structures Single Structure Username C Delete Password Log In Permission to access Data Processing must first be configured on the server Please see the Data Processing documentation on how to grant access Manfred Hendlich 1994 1999 and Cambridge Crystallographic Data Centre 1999 2011 e Itis necessary to obtain permissions to upload and delete structures see Obtaining permissions to upload and delete structures Secti
303. xample of such a match However if you return to the spreadsheet file tutorial csv that you prepared earlier and plot both the protein homology vs number of PC matches and also the cavity homology against the same x axis you should obtain something like the plot below Clearly apart from the obvious existence of a cluster of entries 100 homologous with 1bxo nothing much can be inferred from the protein homology data but the cavity data does reflect the equally obvious correlation between cavity homology and PC matches with the query active site You should remember that for this specific CavBase search you have already demonstrated that about half the pairs that were saved those with scores below the scree transition point Normalised Score 27 8 are not meaningful matches Homology vs No of PC matches 100 t S32 6 t EE 80 o ARa E E oo o 60 z s p a 98 o Cav homology a4 A 4 s E FE Prot homology 40 io 4 ROR REG ewe a TERET watii TETEE steeggsapetesseabibess 20 oe g sd See ee eo T BES T E Coe Se a Ms ew eo 0 r e a e o e a 0 5 10 15 20 25 30 36 40 number of PC matches e Bearing in mind this last observation the cavity data are replotted below with these points removed and a couple of other features have been highlighted e It might seem initially worrying that cavities extracted from structures 100 homologous with the quer
304. y protein are not necessarily 100 homologous themselves with the query cavity 56 Relibase User Guide Cavity homology vs no of matches only observations above the scree transition point Norm Score gt 27 8 100 4 p Ma I A Entries completely homologous with query 1bxo i i o l l ag i I 80 1 T sel ital cae Je e ake am ai NET ERTS i gt P L 60 o 5 MEF E g Z Significant Region of primary interest o maiches E ns eee 40 an o o O I oo E 6 6 8 6 T O E O O bi So Oou b gt o o e o oe 86 20 a 7 pn a oe 08 gt o N a D 4 T T T T T T T 5 10 15 20 25 30 35 40 No of PC matches e But the Ligsite software used to identify and extract the ligand free cavities may find that even though the full proteins may be completely homologous when entries containing different ligands occasionally even identical ligands bound to different chains in the same oligomeric entry are measured the resulting cavities are not always bounded by the same number or identity of residues Thus in the example shown at the end of this section the cavity extracted from the penicillopepsin 2web does not contain the residues ASN8 ILE18 THR19 or ASN117 although it does include SER289 unlike the query site and in this case the CavBase cavity homology calculation will record the absence of these four residue match
305. ying Constraints Section 12 page 144 Run the search see Running a Search Section 6 8 page 74 6 6 3 Hints for Nonbonded Interaction Searching When searching for nonbonded protein ligand interactions at least one of the substructures must be of the molecule type Ligand At least one of the distance constraints must involve a Ligand atom When searching for nonbonded protein protein interactions ensure that the molecule type is set to Protein for each substructure All atoms in a given substructure 1 e a part of the query linked by covalent bonds must be of the same Molecule type Water can be included in any nonbonded interaction search Multiple distance constraints are treated as logical AND operators If you define two distance constraints e g between a Ligand substructure and a Protein substructure both have to be fulfilled simultaneously All required geometric parameters must be defined in the drawing window when the substructure is drawn They cannot be defined after a search has been run If you have drawn a complicated query e g with multiple distance constraints the search may be slow To check whether the hits you retrieve are of interest you can interrupt the search after it has found a few hits and to inspect the hits found thus far The order the contact atoms are drawn in is important you may obtain different search results depending on which atom is drawn first For example the search for a contact between an

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