Lead-Finder user manual and tutorial
Contents
1. Configuration Options for Docking You can set up Lead Finder tasks by specifying parameters in a par file par file is a human readable text file containing references to ligand and protein structure instructions to energy grid map calculations and optional preparation of protein structure The order of parameters in the parameter file is arbitrary Single line comments are allowed after the semi colon symbol Ligand file ligand filename Optional parameter no default value Example ligand ligand mol Specifies name of a file in mol sdf mol2 pdb or gro format containing ligand 3D coordinates When Lead Finder is run with li as a command line option this line of par file is ignored and the file specified after li is used instead ligand_ reference filename Optional parameter no default value Example ligand_reference ligand_reference pdb Specifies name of a file containing such reference ligand from PDB or other source that is positioned in the same binding site of the same protein and in the same coordinate system This option is useful when you already have at least one resolved protein ligand structure and want to dock new ligands to the same protein s site The reference structure is then used to determine the center and the size of energy grid maps If the reference ligand coincides with the docked ligand which may be the case when checking program s performance on experimentally resolved structure2. mode dG and the structures of protein and ligand from the complex should be provided in separate files The folder Example 2 contains a 3D structure of HIV 1 protease PDB 1HSG in protein gro and a structure of the ligand in ligand mol The coordinates of protein s and ligand s heavy atoms were taken from PDB and necessary hydrogen atoms were added As a first step Lead Finder calculates energy grid maps Then it performs optimization of ligand s position and calculates free energy of ligand binding The file 1hsg par contains parameters for the free energy of binding calculations ligand ligand mol the ligand ligand_reference ligand mol the reference ligand receptor protein gro the protein structure exclude_water yes remove water molecules will from the protein structure grid_center reference place the grid center in the geometric center of the reference ligand grid_size reference determine the grid size by the reference ligand grid_atom_types auto calculate grid maps for all types of atoms present in ligand mol Start calculations of free energy of binding with the following command line Lead_ finder f 1hsg par mode dG The free energy of binding and its decomposition to different terms will be saved in text log file The experimental value of the free energy of binding is 13 2 kcal mol Example 3 Ligand docking with pre calculated grid maps When energy grid maps are available were cal
3. be used for 3D optimization of 2D structures ACD pKa software Hwww acdlabs comH can be used for predicting pKa and protonation of ionogenic groups Lead Finder v 1 1 10 User Manual Installation Once the Lead Finder software license is obtained you get the installation distributive and the HASP key USB device that manages your license HASP device stores information about your license so when it comes off the license HASP device blocks the program exploitation Updating license is performed via a simple patch file described below The process of software installation under Windows and Linux is provided below Installation under Windows Install the HASP drivers by executing either haspdinst exe or HASPUserSetup exe files located in Drivers directory of your installation distributive Both haspdinst exe and HASPUserSetup exe recognize your operating system and install the correct driver to the required location The difference between haspdinst exe and HASPUserSetup exe is that the former is a command line application while the latter has a graphical user interface After you have installed the HASP driver copy files lead_finder exe dock_ff lib and residue lib from the Program directory of your installation distributive to the directory where you d like to install Lead Finder Edit your PATH environment variable by adding your installation path to it Now software is ready for use Do not forget to keep H
4. from the Program directory of your installation distributive to the directory usr local bin Please make sure that ownership and access permissions are properly set up Importantly that under Linux you must have aksusbd daemon launched before running Lead Finder software Aksusbd daemon is launched with the following command lt path gt aksusbd Contact your system administrator for technical questions Do not forget to keep HASP key inserted when running Lead Finder Please note that you will need to install HASP LM in order to communicate with your network HASP key Please refer to HASP HL manual for the details Administrator root privileges are required for installation Additional information on particular drivers for different Linux distributions can be found in readme txt file in the Drivers directory Lead Finder v 1 1 10 User Manual Updating software license To update your license insert HASP key and run program update_key The program will prompt you to retrieve HASP key information i or to update the key u Choose option i this will generate client to vendor file which contains information about the current status of your license Send this file to us by e mail and you ll receive file vendor to client which contains updated license information Run program update_key choose option u this time and select your updated file Now your license is renewed Lead Finder v 1 1 10 User Manual
5. ligand to the protein This parameter is optional specifying A and B will be enough in principle but highly recommended since when ligand coordinates are arbitrary which is a common case specified bond length will position ligand correctly with respect to protein Ignoring X is valid only when ligand coordinates are already compatible with covalent bond constraints Lead Finder v 1 1 10 User Manual Protein file receptor filename Optional parameter no default value Example receptor protein pdb Specify name of a file in pdb gro mol2 formats containing protein coordinates When Lead Finder is run with mm as a command line option this line of par file is ignored and the file specified after mm is used instead Atom names of amino acid residues cofactors metal ions etc listed in protein coordinate file must correspond to standard atom names contained in dock_ff lib library file exclude_ water yes no Optional parameter default value is yes Example exclude_water no Specify this option to delete water molecules from protein structure file or retain them during docking calculations This option can be useful when structurally conserved water molecule s is are necessary for ligand binding metal AB Optional parameter no default value Example metal 30 6 When automatic determination of a metal coordination number is ambiguous it may be useful to explicitly define the metal coordination number A is
6. ASP key inserted when running Lead Finder Installation under Linux Install the HASP driver aksusbd daemon either from rpm package or by running installation script RPM packages corresponding to various Linux distributions are provided in the Drivers directory of your installation distributive You can find there packages specially designed for SuSe and RedHat distributions however to our experience they will work for other RedHat and Debian compatible Linux distributions Also the latest versions of HASP drivers for various Linux distributions are freely available at http www hasp com downloads Alternatively you may launch dinst script provided in the directory setup aksusbd This script designed by HASP suppliers will automatically install the optimal driver for your operating system This script also sets up the aksusb daemon to start on boot After you have installed HASP driver you need to have HASP device mounted properly On newer Linux distributions SuSe 8 0 and higher RedHat 9 and higher it is mounted automatically upon HASP driver installation You can ensure if the device was mounted by typing bash c if e proc bus usb then echo 1 else echo 0 fi and examining the output If the HASP device was not mounted automatically type the following string to mount it mount t usbdevfs none proc bus usb To install Lead Finder software copy files lead_finder dock_ff lib and residue lib
7. DB entry 1B5J set of decoy ligands directory decoys set of true albumin ligands directory ligands parameter file screen par and a Perl script do_screen pl for Linux or do_screen_win pl for Windows which launches grid calculations and successive docking of all ligands Results of screening can be processed by another script and gathered in a single file stats txt which can be analyzed with standard table processing software say MS Excel Fraction of actives 0 T T T T T T T T T i 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 Fraction of decoys Enrichment curve for virtual screening of human serum albumin ligands X axis represents the fraction of active albumin ligands known to bind with albumin with good affinity Y axis represents the fraction of decoy ligands Each point on the curve reveals the ratio of true positives active ligands to false positives decoys at the certain fraction of the library screened Totally 16 active ligands and 84 decoys randomly chosen from the set of drug like compounds were used for screening Results show that in the list of compounds ranged by their calculated affinity only one decoy compound occupies 14 th place while active ligands occupy 1 13 and 15 17 places ROC integral parameter describing the accuracy of virtual screening equals 0 9987 which is excellent result g ROC receiver operating characteristic corresponds to the area under the normalized enrichment
8. Lead Fi nder v 1 1 10 Software for Drug Discovery User Manual Modeling the HIV 1 protease complex with U100313 is a particularly tough case for docking software Lead Finder successfully predicts correct ligand position rendered in licorice with 1 2A RMSD as evidenced from X ray structure electron density rendered in wireframe Lead Finder v 1 1 10 User Manual Table of contents Data flow in Lead Finder sssssssssssrrrrrrrrrrrrnrrrnsnrnnnunnnnnnnnnnnnnnnnnrrrnnrnnnnna 3 Protein Structure And Its Preparation For Docking cecceeeeeeeeeeeeee eens eeees 4 Ligand Structure And Its Preparation For DOCKING cececeeeeeeeeeeeeeeeeenneeees 5 TIVSES ATION ie srata statis atteaare aunt ea eis a tanec easton OE Sa 6 Configuration Options for DOCKING lt s2ccicsccendvsaceas ceageasetepeis siadesdziayebaneieleaies 8 Command Line OptiOnS yy sties iat teeta uve lsoes estan aiaets i eeutine aeecgnmetediisakes 11 Example 1 Calculating energy grid MAPS c cece ceceee cece teense eenaaeneeaaes 12 Example 1 Calculating energy grid MAPS ccccce cece eee ee ee eeaeeeenaaeneeaaes 12 Example 2 Calculating free energy Of binding cccce cece eeeee eee eaneneeaaes 13 Example 3 Ligand docking with pre calculated grid MapS s ssssssssssssss s 13 Example 4 Virtual SGreeninG s 2ctiestenduntdewatcctiwinarveitecdsaieieetel ie pede as 14 Example 5 Preparation of trypsin structure for docking ccceeeeeeeee
9. ate file ligand_reference pdb This enzyme contains catalytically important cofactor heme which is essential for cytochrome substrate binding and must be retained for docking Add hydrogen atoms to protein as it was described earlier Add all hydrogen atoms to ligand as it was described above Use standard docking parameter file as described in previous examples Launch docking with key task scr standing for fast screening regime Lead_finder f 5cpp par o 5cpp_solution pdb I 5cpp_energy log task scr 8 You may want to compare Lead Finder docking accuracy achieved with different settings of docking search algorithm Launch docking with different key task values and compare accuracy of structure prediction and speed of calculations as it was described in Example ay 10 The following results were achieved by us using AMD Athlon XP 1 8 GHz with 2 GB RAM screening mode 1 16 A RMSD 0 9 s CPU time docking mode 0 60 A 2s Lead Finder v 1 1 10 User Manual 19 Glossary Binding affinity free energy of protein ligand binding kcal mol dG score Lead Finder estimation of binding affinity Docking positioning of the ligand in the protein binding site with respect to optimal free energy of binding For details see Technology section of Lead Finder internet site Energy grid 3D function placing in accordance a point in Cartesian space and energy of particular type of interaction which would atom of particular
10. culated earlier they can be directly used for docking without recalculating them For this purpose key g in the command line should be used to point to the grid map file Folder Example 3 contains streptavidin 3D structure protein gro ready for docking grid map file lsre map biotin structure ligand mol and parameter file 1sre par described in Examplel Docking with pre calculated grid can be launched with the string Lead_ finder f 1lsre par task dock g 1sre map Calculated ligand poses will be stored in solution pdb file energies in solution log Lead Finder v 1 1 10 User Manual 14 Example 4 Virtual screening Lead Finder can be used for docking a big number of ligands into single protein thus acting as in silico analogue of high throughput screening To achieve the fastest docking with Lead Finder key task scr should be used in the command line To perform virtual screening energy grid maps should be calculated first Then depending on the configuration of your computational resource PC a cluster of PCs etc successive or parallel execution of Lead Finder should be applied to all ligands of interest This can be done in many ways for example using Perl scripting consult your system administrator how to organize screening in your case Folder Example 4 contains all necessary files to perform virtual screening in a successive mode on a single PC 3D structure of human serum albumin protein gro prepared from P
11. curve in coordinates fraction of found true active compounds from 0 to 1 vs fraction of decoys from 0 to 1 Ideal curve reflecting 100 of true actives found at 0 of decoys gives ROC 1 00 Lead Finder v 1 1 10 User Manual 15 Example 5 Preparation of trypsin structure for docking This example illustrates the necessary steps of protein and ligand structure preparation for docking 1 Download trypsin structure 1TNL from PDB at www rcsb org 2 Cut the ligand s trans 2 phenylcyclopropylamine coordinates with any text editor and save it as a separate file ligand_reference pdb 3 As far as a number of protein residues for which alternative protonation states may take place appear in the ligand s proximity special care should be taken while adding hydrogen atoms to PDB structure For this purpose you may launch Model_build program supplied with Lead Finder distribution Model_ build f 1tnl pdb o protein gro ph 7 Next ligand structure should be prepared for docking For this purpose all hydrogen atoms must be added to ligand ACD software for example may be used for adding all hydrogens Note that correct ionization of the ligand must be performed this can be done with ACD pKa module In principle any third party software may be used that can correctly add hydrogens to the ligand 4 Docking may be launched with the string Lead_finder f 1tnl par o 1tnl_solution pdb I 1tnl_energy log Coordinates of found soluti
12. enes 15 Example 6 Preparation of progesterone structure for docking sesse 16 Example 7 Preparation of methanol dehydrogenase structure for docking ee E E ee eee ee er heen en ter ee rt eee renee 17 Example 8 Docking to cytochrome p450 sssssssssssssssrrrrrsssrrnrrrrsnrnrereens 18 GIOSSARY orria teins ee useee aed cae ees aad aes eee ee a ee 19 Lead Finder v 1 1 10 User Manual Data flow in Lead Finder Lead Finder software is a command line application that is available for Windows and Linux platforms Lead Finder takes 3D coordinates of protein and ligand as input along with a short text file containing calculation parameters and global settings Lead Finder can be used to perform protein structure preparation high precision protein ligand docking estimation of the free energy of protein ligand binding virtual ligand screening to search for potent binders for a given protein target Protein structure Ligand structure 3D coordinates of a protein 3D coordinates of a ligand File formats pdb mol gro mol2 File formats mol sdf mol2 pdb gro Source PDB in house structures Source PDB CCDC in house molecular modeling databases molecular modeling Parameter file Parameters to specify calculations File format text file 3 Binding energy calculation D ai D ai Energy estimations Docked structure Free energy of ligand binding virtual 3D coordinates of rank ordered ligand screeni
13. g docking check verbose mode check the ligand and protein structure at input and diagnose problems dG calculate free energies of binding h Print help for using command line options V Display information on structure preparation and docking while calculations are in progress debug Dump of intermediate results which can be sent to Lead Finder developers for maintenance purposes Lead Finder v 1 1 10 User Manual 12 Example 1 Calculating energy grid maps Lead Finder may be used to calculate energy grid maps only without doing docking or other tasks This function is useful when you want to calculate grid maps and store them for future use with other docking experiments thereby reducing the amount of processing and speeding up docking calculations The folder Example 1 contains a 3D structure of streptavidin PDB 1SRE in protein gro and model settings for energy grid calculations in 1lsre par The settings in lsre par are described below receptor protein gro the protein structure will be taken from protein gro file exclude_ water yes water molecules will be removed from the protein structure grid_center 33 13 6 set the grid center at 33 13 6 XYZ coordinates in A grid_size 151515 the grid will span 15 A in each direction grid_atom_types common grid maps will be calculated for all types of atoms reorient_grid no do not reorient grid to enable visualization of a ligand with the p
14. in ligand mol 8 Launch docking with the most precise settings of search algorithm with key task dock standing for the default docking regime Lead_finder f 4aah par o 4aah_solution pdb I 4aah_energy log task dock Coordinates of found solutions will be written in 4aah_solution pdb fileand energies in 4aah_energy log 9 You may want to compare Lead Finder docking accuracy achieved with different settings of docking search algorithm Launch docking using already generated structure grid and parameter files but with different values of key task equal dock default docking settings scr fast screening mode 10 Compare accuracy of structure prediction RMSD is calculated in 4aah_energy log file for each pose when reference ligand is provided and speed of calculations CPU time is also provided in 4aah_energy log The following results were achieved by us using AMD Athlon XP 1 8 GHz with 2 GB RAM screening mode 0 74 A RMSD 2 s CPU time docking mode 0 70 A 5s Lead Finder v 1 1 10 User Manual 18 Example 8 Docking to cytochrome p450 This example outlines the necessity of careful handling with target protein structure which may need to retain cofactor molecules essential for catalytic competency of a protein 1 2 So Download cytochrome p450cam structure S5CPP from PDB at www rcsb org Cut the ligand s adamantanone coordinates with any text editor from pdb file and save it as a separ
15. mino acids optimizing positions of hydrogens etc For this purpose special program called Model_ build is included in Lead Finder distribution Model_build accounts for electrostatic Van der Waals and hydrogen bonding energy when adding hydrogen atoms to a protein and optimizing their positions thereby performing high quality automatic structure preparation Description of electrostatic calculations and optimization algorithms implemented in Model_build can be found in Technology and Bencmarking sections of Lead Finder internet site e Special care should be taken with respect to the defects in experimentally resolved protein structures such as missed atoms or residues incorrect bond lengths angles etc especially in the proximity to the ligand s binding site Model_build automatically repairs some of the widespread defects like incorrect aminoacid labels missing or unresolved side chains of aminoacids e Protein cofactors important for ligand binding should be retained within the protein structure Model_build automatically adds hydrogen atoms to cofactors e Any non intrinsic parts of a protein such as ligand water molecules buffer ions etc should be removed before docking calculations Cofactors and structurally or catalytically important metal ions bound to protein should be retained sometimes conservative structural water molecule s known to play crucial role in ligand binding may be retained as well 1 Format converting pr
16. nd due to this obstacle mol file contains wrong double bonds You must correct bond orders before adding hydrogens This can be done in ACD ChemSketch or other proper software via graphical menu 7 When bonds in the mol file are corrected hydrogen atoms must be added to the ligand This can be done as described in Example 5 8 When protein and ligand structures are ready docking parameter file should be provided by analogy with Example 5 and docking launched with the string Lead_ finder f 1a28 par o 1a28_solution pdb I 1a28_energy log Coordinates of solutions will be written in 1a28_solution pdb file and energies in 1a28_energy log Superposition of deficient ligand progesterone structure extracted from PDB white and energy optimized structure cyan Seemingly small differences of the overall ligand geometry almost all bond lengths in PDB structure are distorted by 0 2 0 4 A influence docking precision Lead Finder v 1 1 10 User Manual 17 Example 7 Preparation of methanol dehydrogenase structure for docking This example outlines the necessity of careful handling with target protein structure which may need to retain non protein residues metal ions water etc essential for catalytic competency of a protein 1 Download methanol dehydrogenase structure 4AAH from PDB at www rcsb org 2 Cut the ligand s pyrroloquinoline quinone coordinates with any text editor from pdb file and save it as a sepa
17. ng score detailed output of binding pose s energy components for all docked File format pdb mol sdf ligand poses File format text file Lead Finder v 1 1 10 User Manual Protein Structure And Its Preparation For Docking e Common file formats such as pdb gro mol2 containing 3D coordinates of heavy atoms and at least functional hydrogen atoms such as ones attached to N O S are accepted as input for Lead Finder e 3D structure of a protein can be taken from publicly available or in house databases or obtained through molecular modeling techniques Accuracy of 3D protein structure is crucially important for molecular docking applications As a rule high resolution lt 2 5 A X ray crystal structures perform better when alternatives exist e Frequently a protein structure especially if comes from PDB will contain no hydrogen atoms Lead Finder requires appropriate placement of hydrogen atoms in the protein structure At least the functional hydrogens must be placed and the following points must be considered i protonation state of a protein depends on pH ii protonation of His should be reviewed as a special case iii protons should be placed on the fittest atom when alternatives exist such as for chemically equivalent atoms in His Glu Asp iv proton orientation may have to be further optimized e Lead Finder can automatically prepare protein structure file by adding hydrogen atoms selecting ionization states of a
18. nts and grid_size parameters are mutually exclusive Lead Finder v 1 1 10 User Manual 10 grid_atom_types auto common lt individual atoms gt Optional parameter default value is auto Example grid_atom_types auto Specify atoms for which grid maps will be calculated auto automatic extraction of atoms from ligand structure common all basic atoms C A N NX O S H P F Cl Br I also individual atoms can be specified for example grid_atom_types CAN NX O H Specify atom types with care since during virtual screening of a big library of compounds different ligand atom types may emerge so that it is better to calculate more grids than to lack them during high throughput calculations reorient_grid yes no Optional parameter default value is yes Example reorient_grid yes Specify this parameter to reorient grids to maximally overlap with the binding site as determined by the built in cavity detection algorithm Reorientation allows reduction of the grid size due to more efficient spatial arrangement exclude_ volume V Optional parameter default value is 40 5 the volume of benzene molecule Example exclude_volume 40 5 Do not consider intramural protein cavities with volume less than V in R gt Other docking parameters n_out N Optional parameter default value is 20 Example n_out 20 Specify number of predicted docked positions poses to be output rot_sulfamide yes no Optional
19. ograms such as Obabel freely available at Hhttp openbabel sourceforge net H may be used to convert your protein structure into one of the file formats listed above 2 The quality of a protein structure may be assessed by a number of internet services available at the PDB site Hwww rcsb orgH Lead Finder v 1 1 10 User Manual Ligand Structure And Its Preparation For Docking Common file formats such as pdb sdf mol mol2 gro containing 3D coordinates of heavy atoms and all hydrogen atoms are accepted as input for Lead Finder 3D coordinates of a ligand can be taken from publicly available or in house databases or obtained through molecular modeling techniques 2D structures of ligands must be converted to 3D coordinates before docking Ligand structure must have all hydrogen atoms in correct places Correct protonation must be performed before docking with Lead Finder A single sdf file containing coordinates for multiple compounds may be used with Lead Finder In this case an additional brief file listing ligand names and corresponding binding energies dG and VS score will be produced for user convenience Crude 2D structure of a ligand 2D structure with all H atoms Complete 3D structure and ionization states adjusted to pH 7 Note that the hydroxyl group bound to the nitrogen atom is ionized 3 ACD ChemSketch software Hwww acdlabs comH or Corina Hhttp www molecular networks com software corina index htmlH can
20. ons will be written in 1tnl_ solution pdb file and energies in 1tnl_energy log Lead Finder v 1 1 10 User Manual 16 Example 6 Preparation of progesterone structure for docking This example outlines the necessity of careful handling with structures from PDB which may have experimental defects like unresolved amino acid residues in protein or distorted ligand s geometry 1 Download progesterone receptor structure 1A28 from PDB at www rcsb org 2 Cut the ligand s progesterone coordinates with any text editor from pdb file and save it as a separate file ligand_reference pdb Note that pdb file contains two identical protein chains each having bound ligand Thus delete one chain with its ligand from the original pdb file 3 In 1A28 structure some of the residues are not resolved experimentally You can build them with freely available Swiss PDB Viewer program or other proper software Save structure with added residues in a separate file 1a28 corrected pdb 4 As far as a number of protein residues for which alternative protonation states may take place appear in the ligand s proximity special care should be taken in adding hydrogen atoms to PDB structure You may use Model_build program for this purpose as described in Example 5 5 Next convert ligand s coordinates to mol file as it was described in Example 5 6 Pay attention to the fact that ligand s geometry in 1A28 structure was initially distorted a
21. parameter default value is no Example rot_sulfamide yes Turn on off the rotation of sulfamide bond during docking rot_amide yes no Optional parameter default value is no Example rot_amide yes Turn on off the rotation of amide bond during docking rot_conjugated yes no Optional parameter default value is no Example rot_conjugated no Turn on off the rotation of conjugated double or aromatic bonds during docking environment solution membrane Optional parameter default value is solution Example environment membrane Choose the type of environment surrounding the ligand binding site Choose membrane when ligand binds to the membrane buried protein site in this case specially adjusted settings of electrostatic calculations and other energy terms will be activated Default environment is solution 7 A is aromatic carbon NX is a nitrogen that doesn t form H bonds for example an amide nitrogen Lead Finder v 1 1 10 User Manual 11 Command Line Options You can use command line parameters to set up Lead Finder tasks and override some configuration options specified in a par file The following command line parameters are accepted f file par The docking parameter file Default docking settings will be used if this parameter is not specified See for more details li ligand mol The ligand structure in a mol sdf mol2 pdb or gro file mm protein pdb The macromolecule protein s
22. rate file ligand_reference pdb Delete unnecessary protein chains B and D from the original pdb file 3 This enzyme contains catalytically important calcium ion and water molecule residue number 785 which should be retained in the structure while other water molecules may be deleted from pdb file 4 Add hydrogen atoms to protein as it was described earlier Note that orientation of hydrogen atoms in water molecule bound to calcium should be correct Correction of water orientation can be performed for example with freely available VMD software 5 Add all hydrogen atoms to ligand as it was described earlier 6 When protein and ligand structure files are ready remember that we want to retain catalytically important water molecule for docking so chose exclude_water no in par file 7 To demonstrate flexibility of grid maps definition in Lead Finder try setting the grid center explicitly via three Cartesian coordinates Par file will look like ligand ligand mol ligand will be taken from ligand mol ligand_reference ligand_reference pdb reference ligand will be taken from ligand pdb receptor protein gro protein will be taken from protein gro exclude_ water no water will not be excluded grid_center 15 1915 XYZ coordinates of grid center grid_size reference grid size will be determined by reference ligand grid_atom_types auto grid maps will be calculated for all types of atoms present
23. rotein structure Start grid calculations with the following command line Lead_ finder f lsre par mode grid og 1lsre map The calculated energy grid maps will be saved in 1sre map file Note that map files are loaded to memory sufficiently slower than bin files thus for re using calculated energy grid maps in future docking experiments it is practical to save them as bin files The advantage of map files map is AutoDock file format is that they can be visualized with freely available VMD software Also this option may be useful in qualitative analysis of ligand binding forces This is an example of visualization of grid map files with VMD software The structure of streptavidin 1SRE is rendered in new cartoon reference ligand 2 4 hydroxyphenyl azo benzoic acid in licorice VdW grid in gray wireframe H bond donor grid in blue wireframe The reference ligand s shape seems to fit well into the protein s binding site and the ligand s carboxylic group is placed correctly for H bonding interactions Other grid maps electrostatic etc may be loaded to VMD and visualized as well Lead Finder v 1 1 10 User Manual 13 Example 2 Calculating free energy of binding Lead Finder can be used to calculate free energy AG of ligand binding to protein when structure of the protein ligand complex is available from X ray NMR molecular modeling studies etc For this purpose the program should be launched in
24. s RMSD between the reference and predicted ligand poses will be calculated and saved in a log file Note that reference ligand does not necessarily be a chemically consistent structure when actual reference ligand is missing you can take some points in the ligand binding site and write them into reference ligand structure say in pdb format this may help in determining overall placement of docking solutions filenamel filename2 additional_ reference additional_ reference Optional parameter no default value Example additional reference ligand_referencel pdb Specifies coordinates of additional reference ligands if needed Additional reference ligands can be used for adequate RMSD calculations of docked ligand pose and reference ligand when a number of symmetric ligand binding positions are valid In such case when a number of corresponding symmetry related reference ligand positions are provided RMSD will be calculated with respect to the nearest reference Anyway accuracy of ligand docking does not depend on additional reference ligands covalent AB X Optional parameter no default value Example covalent 2 2542 1 542 When ligand is covalently bound to a protein numbers of bonded atoms must be provided Specify A as the ligand s bonded atom number and B as the protein s bonded atom number The enumeration for ligand and protein starts from zero Additional parameter X specifies covalent bond length linking
25. the metal atomic number enumeration starts from zero B is the metal coordination number that is used in the calculation of a directional potential of metal ligand interactions When there are several metal ions in the ligand binding site each ion can be described by its own string Energy grid maps grid_center reference X Y Z Required parameter default value is reference Example grid_center 13 5 46 3 10 4 Choose one of the two methods for grid center placement reference center at the reference ligand if one is used Alternatively specify three Cartesian coordinates in X Y and Z in grid_spacing X Required parameter default and recommended value is 0 375 Example grid_spacing 0 375 Distance between neighboring grid points along particular Cartesian direction in A grid_size reference X ABC Required parameter no default value Example grid_size 10 10 10 Choose one of the two methods for grid size definition 1 reference a grid with X A margin in each dimension from the reference ligand default value of margin is 6 A 2 an explicit definition grid_size A B C with three dimensions lengths of the grid box edges in A Alternatively grid size can be defined by setting the number of grid points with grid_npoints parameter instead grid_npoints ABC Optional parameter no default value Example grid_npoints 120 60 65 Specify grid size in a number of grid points in each dimension grid_npoi
26. tructure in a pdb gro mol2 file og grid bin Calculate and save grid maps after docking The acceptable grid file formats are bin binary format and map human readable format Grid files with map extension can be visualized with VMD software but they are larger in size and are slower to load than the binary files g grid bin Load grid maps from file and skip grid map calculations o solutions pdb Output 3D coordinates pdb of docked poses os brief_ output log Output only ligand s name and calculated energy dG and VS score into a text file This option may be useful when docking many ligands from an sdf file omm protein pdb Output a protein structure file in pdb gro mol2 formats The binary file with protein structure can be used subsequently as input for docking as it allows faster processing I solutions log Output free energy of ligand binding for each docked pose in a human readable log file task dock scr Specify one of the two available lists of settings for the conformational search algorithm docking dock or virtual screening scr Settings are optimized with respect to docking speed robustness with screening being the fastest and docking the most precise details can be found in the Technology section of Lead Finder internet site mode norm grid check dG Choose one of the following modes of operation norm run docking calculations grid calculate energy grid maps without doin
27. type possess being placed at this point Correspondingly grids are calculated for each type of interactions and each atom type HASP key USB device that controls your software license Virtual screening rank ordering of compounds from particular library according to estimated binding potency with respect to particular protein Lead Finder docks each compound from the library and calculates VS score for docked ligand which serves a quantitative measure of ligand activity binding potency VS score Lead Finder estimation of ligand activity binding potency for rank ordering ligands during virtual screening For details see Technology section of Lead Finder internet site Lead Finder v 1 1 10 User Manual
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