PDF manual - FoldX plugin for YASARA
Contents
1. Version 1 4 21 User manual Table of contents PECTACS EOE A E E E E E E E AEE E TEE 3 losta laton and EST LG norrena E E E 4 S eO r T T A E er eer ret re 4 ap Bc Cas e T E E E ENE E T OE D TEE E E EOE E E E E E PA 5 Ce pE T E E T 6 F ldX tools aY ASARA reran t N EAE O EAEN A aE AI 7 Pr OO a A A 7 Contex TNE US sanee a E E E an dia hee as 8 FOV ACCROSS anono eer r E ous tous ctsanes acs TE onto aatnomnenseaeoss 9 CATO ALI OT SCS ed ihaaa Sie tele E E EN 9 Protein DNA nucleotides and metals recognized by Fold X cc cccceeseeeeeeeeeeees 10 a 101 0 E ae ee ee Renn en E eee ean een ee eee ene 12 B ects Wich duU hc mre ey et a Ae Ree 13 ROE 1D faeces scence a chee tle mes cas Rats aia ase ei tree 13 SADNE OLON ole eee Reese ee mre arate eaten niente a a art ae 14 Alanne scan OF OD CCl aichatusatech E T eat aaanets 15 Enere y analysis Oi ODJECE E EE 15 Interaction enere y Ol MOE CUIES jaicteeer el a leas eater 16 Muta POSIOQUIC outeni e aE A ual OT A aa eee 17 Mu tate Muli Diet CSICUCS sstetccltyuvevedsinn A EE E A eee aeeeunes 22 Build momol y MIO less ag ose a a a a eee eat ee ee 25 DAVE FAST CAICUTATI OI nesre oSucioasictoe aces aie AA nE O a Ea EA EAA EER A e AEA AEO 26 ConneuropPiUE isses 26 References to the FoldX methodology 0 0 ccccccccssssessseeeeeeeceeeeeeeeeeeeeeaaaaeeeeeeeeeeess 2a UP ANTVATN SS CT CIS CS sts otic eceeniesicisst ese icephes cic ug otivinv enc s oc ste stoo oso cacao venison ese 28 Introductio M2. 1 Schymkowitz J Borg J Stricher F Nys R Rousseau F Serrano L The FoldX web server an online force field Nucl Acids Res 33 W382 8 2005 2 Schymkowitz J Rousseau F Martins IC Ferkinghoff Borg J Stricher F Serrano L Prediction of water and metal binding sites and their affinities by using the Fold X force field Proc Natl Acad Sci USA 102 10147 52 2005 3 1 Guerois R Nielsen JE Serrano L Predicting changes in the stability of proteins and protein complexes a study of more than 1000 mutations J Mol Biol 320 369 87 2002 28 Training exercises Introduction In this section we will let the FoldX plugin loose on some real world examples and give you step by step instructions on how to proceed and analyze the results We will use the P53 tumor suppressor protein as our example molecule In a first exercise we will show you how to make a point mutation with FoldX and how to determine if the mutation 1s stabilizing or destabilizing for the P53 structure In a second exercise we will design a mutation in the P53 structure at the DNA binding interface and determine how the mutation affects the interaction energy of P53 with the DNA strand Fold X energies Before we start some basic information about FoldX energies 1s necessary First of all FoldX energies are expressed in kcal mol The main focus of FoldX is the prediction of free energy changes e g what happens to the free energy of the protein when we mutate an
3. Y ARI ASP ARG ARG THR Gill ee a a a a ee es m m bj 1 Men r f i ia Hw BR We E ahaa ee SC ENAT rh a eT ST View of the first options menu with Show new and disrupted hydrogen bonds selected r igj Toggle the Visibility between this mutant and the WT structure and see how the hydrogen bonding changes Check the output in the Console to see what the change in interaction energy is between P53 and DNA chain G upon mutation fio Eat Simulates Anike View Efiecs Gpticm Wieda Heip Cp g C k TA ia ws Yeisen mamma se WT ee as Set Aoa We see that the mutation decreases the interaction with DNA strand G by approximately 1 kcal mol we lost 1 hydrogen bond Instead of DNA protein FoldX can of course also calculate interaction energy changes in protein protein or peptide protein complexes 38 Troubleshooting Advance note Whenever you run into a problem or you experience a crash always check first 1f you have carefully followed the installation instructions and you are using the latest version of the plugin and the latest version of YASARA If the problem persists check if the issue 1s listed here If not don t hesitate to contact us Analyze menu does not open As explained in the first use section of this documentation website the Analyze menu only becomes active when a structure is loaded So open a structure with e g Now the Analyze menu is active and a FoldX submenu should be pr
4. the free energy of the wild type WT and the mutant MT and make the difference AAG change AG MT AG WT FoldX is trained to make AAG change approach experimental values It is important to realize that AG WT and AG MT are meaningless numbers as such These do not correlate with experimental values Only AAG change does As a rule of thumb we use AAG change gt 0 the mutation is destabilizing AAG change lt 0 the mutation is stabilizing The error margin of FoldX is approximately 0 5 kcal mol so changes in that range are insignificant Calculation speed The calculations were performed on a Linux pc with an Intel Core Duo2 2 8GHz processor with 3MB of RAM memory Repairing a structure 1s the most computationally intensive procedure as all sidechains have to be re evaluated for their energy This takes on average 2 5 minutes for a protein of about 300 residues One point mutation takes a few seconds for mutating to small amino acids to 1 2 minutes for larger amino acids with 10 more degrees of freedom in their sidechain R K Calculation of interaction energy is a matter of seconds Protein DNA nucleotides and metals recognized by FoldX Below are lists of molecules that are recognized by FoldX This implies that FoldX can include these in the free energy predictions For example it is thus possible to predict the effect of an amino acid mutation on DNA binding or metal binding List of amino acids Name
5. 3 letter 1 letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Phenylalanine Phe F Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Phosphotyrosine Ptr y Phosphothreonine Tpo p Phosphoserine Sep S Sulphotyrosine Tys Z List of DNA nucleotides Name PDB file code 1 letter Adenosine A a Guanine G g Cytosine C c Thymidine T t 6 methylated adenosine 6MA b 5 methylated cytosine SCM d List of nucleotides Name PDB file code Adenosine diphosphate ADP Adenosine triphosphate ATP Guanosine diphosphate GDP Guanosine triphosphate GTP List of metals Name PDB file code Calcium CA Magnesium MG Manganese MN Sodium NA Zinc ZN Iron FE Copper CU Cobalt CO Potassium K Waters Name PDB file code Water HOH 12 Fix residues FoldX commands such as RepairPDB the FoldX energy minimization and BuildModel make mutations or complete models will move residues by default RepairPDB minimizes a structure by rearranging the sidechains to find a new energy minimum BuildModel will rearrange neighbouring residues of a mutation spot to optimize the mutated protein There are cases where the user does not want certain residues to move either during RepairPDB or BuildModel The Fix residues command lets you select resid
6. Ile in 3 runs each Each mutation is loaded as a new object and the energy difference is printed in the console The same results are also listed in a tab delimited text file called FOLDXSUMMARY out in the cache folder This file can be saved in a custom folder together with all other output files using the option Save last calculation All models and repaired structures can be saved as PDB files by clicking for each object separately Or you can save the entire YASARA scene keeping all Objects colors and orientation including superposition by clicking To get the most accurate results we recommend to use the Repair object command first For reference this table lists all residues with their 1 lettercode and 3 lettercode as used in the plugin Name 3 letter 1 letter Alanine Ala Arginine Arg Asparagine Asn Aspartic acid Asp Cysteine Cys QUARK 22 Phenylalanine Phe F Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val Vv Phosphotyrosine Ptr y Phosphothreonine Tpo p Phosphoserine Sep S Sulphotyrosine Tys Z Mutate multiple residues This option does in many ways the same as the Mutate residue option The difference 1s that with this option you can make multiple mutations in one structure such as double triple mutations on more than one residue posit
7. need to do in each menu 33 1 Only select Calculate stability change 2 Select Trp 3 Move neighbours and Show VdW clashes in WT and mutant 4 Don t change any numerical options in the last menu Eanes H diab Pe etait ated vidi Cell eh era wl Ravn regir y mul Epa bi alii Hh fee Sme Grud and eia Freng bones _a THR PRO FRO PRG Y TH ARG YAL ARa AA MET AMA HE TYR Y3 HN SER GAH He T THR HU VA 0 151 152 158 Ti naa 154 Mig bigs l y iah atz kgs ig is a I biy ir ah mt i View of the first options menu with Move neighbours and VdW T selected More information on all these selection menus is explained in detail in the mutate residue section of the guide Analyze the mutation FoldX has mutated the Ala to Trp and the structure with the Trp mutation has been loaded in the next Object 3 and is superposed with the wild type WT Object 2 We selected an option to show the VdW clashes in WT and mutant The atoms that give rise to steric clashes are colored in red Toggle the Visibility of Object 2 WT and Object 3 mutant and see how many clashes we introduced by mutating the Ala to Trp 34 relent spoT 2 NE aan Zoomed in view on the original Alal59 no Vander Waals clashes here Fa a Fi i f rA r Fi Fi j Wine 4 ii Fieiniit HE HE P F Te HEHH A i 7 F 4 i m Tain ririrriris 4 j F BT F d p 1 in 1b
8. one run will be loaded as YASARA Objects Save last calculation This option lets you specify a target folder and or filename prefix for the beginning of all files of the last calculation to be saved A calculation is either a Repair Alanine scan BuildModel Mutate residue Mutate multiple residues Build homology model Interaction energy Stability The first selection window lets you choose whether you want to save all files or just the FOLDXSUMMARY out file This file contains mutation energies and is only created after an alanine scan or a mutation run In the next selection window you can either select a single folder or a folder and filename prefix Folder E g selecting c testrun will save all last calculation files to that folder Folder with filename prefix E g selecting c testrun MyRun will save all last calculation files to the testrun folder and put MyRun_ before every filename In this way it is possible to save more than one calculation in the same folder by using different prefixes Save last calculation saves the Foldx output tales of the last calculation to a user sspecri ied folder with optional filename prefix For more information on the content and format of FoldX output files we refer to the FoldX manual that can be downloaded from foldx crg es Configure plugin See the section Installation and first use for explanation of this option 23 References to the FoldX methodology 1
9. Asp to a Tyr FoldX will then calculate the free energy of the wild type WT and the mutant MT and make the difference AAG change AG MT AG WT FoldX is trained to make AAG change approach experimental values It is important to realize that AG WT and AG MT are meaningless numbers as such These do not correlate with experimental values Only AAG change does As arule of thumb we use AAG change gt 0 the mutation is destabilizing AAG change lt 0 the mutation is stabilizing The error margin of FoldX is approximately 0 5 kcal mol so changes in that range are insignificant Minimizing the structure FoldX assumes that the starting structure has been energy minimized Although crystal structures with high resolution often represent a low energy form FoldX performs best when we minimize it just before we do the predictions This FoldX procedure is called RepairPDB and should be done on each structure you want to perform calculations on 29 Download and open the YASARA scene 2AC0 sce in YASARA This is a part of a tetrameric complex of the transcription factor P53 bound to DNA I removed 3 of the 4 P53 structures for simplicity and visualized some nice features Load the scene with fone hanima A nee Hil 7 F 6 He iomas Syak VRSAAA a WHAT Cty at Sman P030 Alor P53 monomer bound to DNA To Repair or minimize the structure with FoldX go to Fetes haimaa oe A Pea
10. E iE F C T T T T K E gt T OO Hr omde ayi HSA eT Cy at Amon 2050 Alor Hovering the mouse at the bottom of the screen will open the sequence selector Search for Ala 159 in Object 2 not Object 1 32 Per T T f paii i F T p hia FUE i mamap HUHA e F i I p aicha A CAL i a gaa i Bait Sock i i ede nians Hiji belay Hahira F O Hedo iuit b AS lide hydeegen bonds THR PRO FRO PRO GY TH ARG w Sa FI ge TYR LYS GH SEK GM Ha MET THR GU VA Lint 1 182 isa isa Sig Uen E ia ia bo ibs iba a iaa ib ia ii i eji iat T T T E F lks h g E T T T T c c CIG ie jiya EARANN dea amp WHAT IF Oty af Samco S Aleem Zoom in on the Alal59 by keeping the CTRL button pressed and at the same time left click on it in the sequence selector We are going to mutate this residue to the largest natural amino acid tryptophan W Right click again on Ala159 in Object 2 in the sequence selector and go to Esans himan Se oe A ne i al t nmen B THR PRO PRO PRO Y THA ang w Label TA E TYR LYS GIN SER GN He MET THR GU VA si 1 182 isa 14 ga ag ji Uriba i 1a w bbi ibs i ia iaa im ia ii i bj 2 biae T a E pee L g p a T T T g E Her Were 36 HE YS daga a W ITA E ir DH mM Samco 2050 Aleem Right k on Ala159 and select FoldX gt Mutate e to mutate this specific residue A number of menus is now presented and here is what you
11. This will result in a plugin crash This should be fixed in the next FoldX release In the meantime always leave the Move neighbours option checked If you really don t want neighbouring residues to move you can use the Fix residue option to freeze residues manually using Build homology model issues Error The given template sequence in the sequence file was not found in the template Structure If you are sure that the alignment file contains the correct template sequence check the structure for multiple residues with the same residue number e g 1EJG residue 25 If multiple residues with the same residue number are present these additional residues will make the structure extracted template sequence longer and the plugin will raise an error because the structure contains more residues than the provided template sequence Solution 1 Run a FoldX RepairPDB first You can do this manually from Analyze gt FoldX gt Repair object IAlternatively you can check the Repair option in one of the selection menus from the IBuild homology model command IRepairino the structure will only keep the residue that occurs first in the PDB file and delete the next one s I psolution 2 Manually delete the additional residues so that each residue number occurs only once 40 Author and contact support The FoldX plugin for YASARA was developed and is being maintained by Joost Van Durme PhD at the VIB Switch Laboratory Vrije Univers
12. cts All of these will be structurally aligned superposition using the Mustang algorithm for direct comparison The requested output stability change interaction energy change will be plotted in the YASARA console The same results are also listed in a tab delimited text file called FOLDXSUMMARY out in the cache folder This file can be saved in a custom folder together with all other output files using the option Save last calculation All models and repaired structures can be saved as PDB files by clicking for each object separately Or you can save the entire YASARA scene keeping all Objects colors and orientation including superposition by clicking To get the most accurate results we recommend to use the Repair object command first 25 Build homology model When a homology modeling task 1s desired or the number of mutations to make is too large for the option Mutate multiple residues the Build homology model option is your choice for the job o Build homology model will run FoldX BuildModel to model a given target sequence on a Selected template structure object When this option is selected the following menus are presented Object selection menu A single object that serves as the template structure can be selected here Select alignment file The alignment file is a plain text file containing only two lines The first line represents the sequence of the template protein This can also be just a part of the t
13. e latest version FoldX 3 Beta 5 expires in 2012 from http foldx crg es after free and simple registration for academic users Commercial users should contact CRG tech transfer to acquire a license A trial version can be obtained first Install FoldX by unzipping the file you just downloaded This will unzip two files an executable file and a file called rotabase txt Both are necessary 3 Download and install Python On Linux and MacOSX machines Python is installed by default Windows users can download Python from http www python org download or install it from 4 Download and install the FoldX plugin for YASARA The plugin can be downloaded HERE The content of the plugin has to be extracted in the yasara p lg folder or yasara plg folder for Windows users After doing that you should have these 8 files in your yasara plg folder 1 foldx py 2 foldxaminoacids txt 3 foldxanalysecomplex py 4 foldxbuildmodel py 5 foldxplotoutput py 6 foldxrepair py 7 foldxstability py 8 foldx cnf First use Start YASARA and load a structure Now go to In the first browser window select the FoldX executable file and in the second browser window select the rotabase txt file that came with FoldX As long as you don t change the location of those files or you don t overwrite the foldx cnf file be careful when unzipping a new version directly in the p g folder this procedure has to be done only once Citing the plugin If y
14. emplate protein as long as this sequence exists in the template structure FoldX will always model in the context of the whole structure so the part of the template that you did not type in the alignment file will still be in the final model The second line holds the target sequence of the protein to be modeled This sequence has to be of the same length as the sequence of the template on the first line and can only contain valid 1 letter amino acid characters Modeling insertions and deletions 1s not yet possible in the current version of FoldX Tip Extract the sequence of the template structure using the YASARA command ListRes or iclick lAnalyze gt List gt Residue to get the exact amino acid sequence of the template structure Copy and paste the template sequence or just the part you want to align from the Console to a text editor on the first line Ihen align it with the target sequence by typing the target sequence on the second line Save the alignment to a file and feed this file to the plugin GSSPKAVALYSFAGEESGDLPFRKGDVITILKKSDSQNDWWTGRVNGREGIFPANYVELV GSS PRAVALYSYAGDDSGDLPFRKGDLIRGLKKSDSQSDNWTGRMNGRKMIFPANYVELV First line contains the template sequence does not have to be the full sequence of the template structure Second line contains the target sequence to be modeled 26 The rest of the menus work in the same way as in the Mutate residue option The resulting homology models more than one if more than
15. esent FoldX menu does not show up You are using an old YASARA version The plugin only works from YASARA 10 11 22 and higher Please upgrade to the latest version You forgot to download and install the plugin It is not enough to install YASARA and FoldX to be able to run FoldX from YASARA You need to download and unzip the plugin zip file from the homepage or HERE Please consult the Installation and first use section for details on installing and unzipping the plugin in the correct location You are using Windows Vista or Windows 7 This workaround comes from the YASARA documentation YASARA does not have write permissions in its own directory Especially Windows Vista 7 has a deep sitting problem dealing with permissions 1 e even if you installed YASARA in the Program Files folder providing all the required authorization Windows may still prevent it from running correctly To fix this issue right click on the yasara folder then click Properties gt Security select group Users click Edit tick Full control Allow click Apply Verify that there is now really a hook in the Allow column for Full control If you do not have the required administrator privileges to perform this operation simply install YASARA in your home directory 39 Unchecking the Move neighbours option results in a crash When Move neighbours is unchecked FoldX does not generate a reference wild type structure as the plugin expects
16. estions The 3D graphics and intuitive interface make it a very user friendly protein and nucleic acid analysis package It 1s also easily extendable by means of plugins FoldX is normally run from the command line without graphics By installing the FoldX plugin for YASARA you can access the most important FoldX tools at the touch of a button in the 3D graphical YASARA interface You directly see the results of your FoldX protein analysis on screen FoldX YASARA and the FoldX plugin for YASARA are all available for Linux MacOSX and Windows This manual is a guide through the installation and usage of the FoldX plugin for YASARA The web manual can be found at http switch vub ac be foldxyasara Have fun Installation and first use Installation 1 Download and install YASARA The FoldX plugin for YASARA can be used in any stage of YASARA View Model Dynamics Structure Twinset YASARA View is the free version of YASARA and can be downloaded from http www yasara org viewdl htm after free and simple registration You will receive a download link in your mailbox shortly after registration Installation instructions are in the mail from YASARA The plugin only works from YASARA version 10 11 22 and higher Upgrading to the latest YASARA is strongly recommended 2 Download and install FoldX The FoldX plugin for YASARA requires that you have FoldX installed on your computer For compatibility purposes you need to download th
17. h Ionic strength of the solution M x 100 The multiplication by 100 was needed for YASARA implementation since no decimal numbers are allowed in menus So the default of 5 is actually 0 05M Van der Waals design When set to 2 it considers rotamer penalizations due to internal clashes maximum penalization for interresidue VanderWaals clashes a ceiling for the VanderWaals clashes between two atoms of 5 kcal mol and strict H bond geometry When set to zero there is a weak rotamer penalization there is a ceiling for the VanderWaals clashes of 1 kcal mol and we use a relaxed H bond geometry This option should be set to 0 when doing an Ala scanning mutagenesis of a protein Whenever doing design or repairing a PDB it should be set to 2 default in the RepairPDB in this plugin T vull For Pik opeecre dail i paren Humbe oa Temperature H Momot ayat Second options menu After the mutation design the Repaired structure 1f selected and all the runs of the mutations will be loaded into YASARA as new Objects All of these will be structurally aligned superposition for direct comparison see screenshot below The requested output stability change interaction energy change will be plotted in the YASARA console see screenshot below The individual energies as well as the average energy over all runs will be printed 21 X ii a wee i N E AN as ee Result of a mutation from Val to Arg and
18. i j i jj i ig j 1 f r ws F e i F i j Pn p n i E pa d 2 a E F P E ET a i a E p 1 gt j we Esans hpisag m if h E a a a r i at S H 1 Es i i a i r _ i e l ii r De agit z el id l j ai n k j fp i a fd all mao ail ae PA vistas Ae i Re S a er a x Eo B a8 d n ra y TTA gT a ok Saale ate Pe Pe y _ x i f dia n E j DE k i d bd ke fe is EH HARE lt lt g ives a e a oe Tin 0 re en OF Span T act ten WATIE gt Zoomed in view on the mutated Ala159Trp lots of red Vander Waals clashes here i CEI Van der Waals clashes are red colored atoms Do you see a difference around the mutation iSite between WT and mutant Toggle the Visibility of WT and l mutant to see the differences Open the Console by pressing the spacebar twice and see the free energy change of the mutation Anything above a change of 0 5kcal mol is already assumed to be destabilizing 35 _ fie Eat Gimusi Ansiyre view Efiects Gini wies Heip C ga i ra f A In the console to open press spacebar twice we see an energy change of 29 kcal mol Clearly this is severely destabilising the structure Predict effect of a point mutation on protein DNA binding Hide Object 3 by toggling its Visibility so that only Object 2 the repaired WT is visible First turn on all atoms in the molecules G and H DNA again as you did
19. ing the spacebar once or twice ee FT F F F F F Y J6 Gyek YOR da FoldX tools menu in YASARA All FoldX tools in YASARA can be accessed by clicking Context menus A subset of the FoldX tools is also accessible via the atom residue and object context menus The atom context menu appears when an atom is selected and the right mouse button is clicked The residue context menu appears when a residue in the sequence selector is right clicked see screenshot below a m Wi MA ARG 3ER ABH FPE ASH VAL CYS ARG IEU O y TH PRO a i 10 Ti i 13 T 18 1 iF 1 18 70 Ti Fri ri Fi Fi H id bl Pi il H l hi T L D D Fi il inosan A Se at Eel T Alo Residue context menu appears when a residue in the sequence selector 1s right clicked This way of accessing some of the FoldX tools has been made to speed up quick analysis as the right clicked atom or residue is automatically selected for analysis When selecting calculations on a YASARA Object through these context menus the selected Object is the one the right clicked atom or residue belongs to The object context menu appears when an object is right clicked in the HUD see screenshot below Obje Bet context menu appears ET a s7 in the HUD is right clicked Fold X energies The main focus of FoldX is the prediction of free energy changes e g what happens to the free energy of the protein when we mutate an Asp to a Tyr FoldX will then calculate
20. ioceno a eee Ta tigi NUT ar Nd Seer EN tenet ey eee Ana ere ee 28 Fold X enero 1G pur aes a cate tae tas ch amedaaeadmancuuce E teeta 28 Minimize NS SIU CUE aisean a ae a AS 28 Predict effect of a point mutation on protein structure stability cc eeeeeeeeeeeeeees 31 PINAY Ze NING AMM OAE E aisaar eine dis wag O 33 Predict effect of a point mutation on protein DNA binding eeseeeeeeeeeeees 35 Trono HOONE ae E T ERC Coan eee a Re ee Ra er 38 INGY GING NO Eao E EO EA EEO 38 Apay Ze MENU COCSMIOU OPC enun a O R O 38 FoldX menu does Not show Up aiiiresirai riens na E EA 38 Unchecking the Move neighbours option results in a crash 0000000oooonneennnennnssssssss 39 Build Homology Modelis sU S seie asan E E A 39 Anthor andcontact Suppor eessen a E EEE TET A E T 40 Preface The FoldX plugin for YASARA is a software package to access and run FoldX commands in YASARA FoldX is a molecular modeling and protein design software program that calculates energy differences that come close to experimental values FoldX can minimize a PDB structure mutate one or more residues to new residues do protein stability analysis protein protein interaction energy analysis and much more FoldX is widely used to predict the effect of mutations on the stability of a protein or to predict the effect on protein protein binding YASARA is a molecular graphics modeling and simulation program that finally makes it really easy to answer your biological qu
21. ion I Mutate multiple residues allows to select multiple residues and indicate the replacing 1 amino acids for multiple mutations in one structure i The main differences in the menus that are presented are Residue selection menu Here you can select multiple residues to mutate with FoldX 23 DH Wiege lia O SSAA ee Le We i Residue selection menu allows to select multiple residues to mutate simultaneously New amino acids In this text box you have to type the new amino acids in sequential order in 1 lettercode as given in the table above E g if you selected in the first menu residues T13 A30 R54 and W67 you indicated that you want to make four simultaneous mutations in the structure In the text box you then type four replacing amino acids e g SLEY This means that following mutations will be made T13S A30L R54E and W67Y Note that the amino acid 1 lettercode is case sensitive since this allows for modified amino acids to be handled by FoldX see the amino acid table above 24 Typi Fih cee biS ec i Re Bete i ST ce ch Flew iiei Gaia Tiii BEB j h m mon HT Aloi In the text box we type in N to C order the replacement residues of those we selected in the previous residue selection menu The rest of the menus work in the same way as in Mutate residue After the mutation design the Repaired structure 1f selected and all the runs of the mutations will be loaded into YASARA as new Obje
22. iteit Brussel Belgium For support write an e mail to joost vandurme switch vib vub be Cheers
23. mmand It determines the interaction energy between 2 selections of Molecules It is allowed to select more than 1 molecule in each selection window E g if you select molecules A and B in the first selection and C in the second selection A and B are joined in one entity AB The interaction energy between AB and C 1s then calculated The results are displayed in the console The way it operates is by unfolding the selected targets and determining the stability of the remaining molecules and then subtracting the sum of the individual energies from the global energy In case there is a metal bound between the two molecules it will assign it to the one which makes the stronger interactions with the metal So clearly Interaction energy of molecules calculates the interaction energy between two selections of Molecules As an extra this command also lists the residues at the interface of the selected molecules in the console feo Edt Serudates Anette new Eregi Option ird nlp A D C TOS cee oe W A Result of interaction energy calculation between joined molecules ABD and molecule C Also the interface residues are printed 17 To get the most accurate results we recommend using the Repair object command first Mutate residue Mutate residue mutates one amino acid residue to one or more new amino acid residues using FoldX BuildModel This command executes the FoldX BuildModel command Subsequently the user is
24. ng al pem nmen oC O O 30 Select the object for repairing And select the only object in the list Repairing a structure can take a few minutes depending on the size of the protein When the Repair is finished the Repaired Object is placed in Object 2 see top right corner and superposed with the original Object 1 Take a look at the sidechains and see what FoldX has done while Repairing If you feel the repair takes too long more than 10 minutes due to a slow computer download and open this YASARA Scene with the Repaired Object Because we will continue working with this Repaired Object we can now hide the entire Object 1 by toggling the Visibility column in the top right corner head up display HUD As repairing can take quite some time it is a good idea to save each repaired structure for re use later on Save the repaired structure as a PDB file with and select the second Object in the leftmost Sequence column 31 Predict effect of a point mutation on protein structure stability Turn on all the atoms in the DNA chain These are chains molecules G and H Show the sidechain of Ala159 in the core of P53 by searching for it in the sequence selector in Object 2 not Object 1 right click on it and go to THR PRO PRO PRG GiY THA ARG WAL AR l a MET ALA HE TYR Y3 HH SER GAH He MET THR U YA mo ti 1818 mh 14 SF i W a ibi ie w H a ja a a e i M IF ey t bia T T T E E CE r E
25. ou publish results that were obtained with the FoldX plugin for YASARA please cite The FoldX plugin Van Durme J Delgado J Stricher F Serrano L Schymkowitz J and Rousseau F IA graphical interface for the FoldxX forcefield Bioinformatics Advance Access published April 19 2011 doi 10 1093 bioinformatics btr254 Krieger E Koraimann G Vriend G Increasing the precision of comparative models with YASARA NOVA a self parameterizing force field Proteins 2002 May 15 47 3 393 402 If you use YASARA for anything else than molecular graphics please consult your YASARA documentation for the proper citation s FoldX tools in YASARA Preface In this section we explain the various FoldX functionalities inside YASARA It is recommended that you have practised with YASARA and FoldX before But don t worry all commands are straightforward and easy to use That s the point of the plugin However we will refer to YASARA specific nomenclature such as Objects and Molecules Their definition can be found in the YASARA documentation on your computer in yasara doc index html Sometimes we will not explain all details on the FoldX commands provided in this plugin but refer to the detailed FoldX manual which can be downloaded at http foldx crg es The output of FoldX calculations is always printed to the YASARA console when the plugin ends The console should open by default but when it fails you can enter the console by press
26. presented with following menus Residue selection menu Here you can select one residue to mutate to other residue types orig yak due selection menu fa He I Resi Select FoldX routines RepairPDB repairs the structure as in Repair object before making the mutation The mutations will be made using this repaired structure Calculate stability change calculates and display the stability change upon mutation So the difference in stability between mutant and wild type structure This predicts whether a mutation will stabilize or destabilize a structure Stabilizing mutations will have a negative value ddG Calculate interaction energy change this option should only be used when the Object contains multiple Molecules It calculates and displays 18 the interaction energy change between any possible pair of Molecules in the Object after mutation Seok Pee ee ae FM Reger Do P Caure plait change ml akoulade andor BAr cree gy change J Her en bya a F Selection menu for FoldX command Note the output will contain a column named corrected interaction energy This is the interaction energy corrected for intrachain clashes See the FoldX manual for more information Select new amino acid residue s here you can select the residues that should replace the original residue Each selected residue will result in a new model made with FoldX 19 Selection menu for new residues Set FoldX o
27. previously because the FoldX run has hidden it it rearranged the view to show the VdW clashes Show the sidechain of Arg273 of Object 2 by searching for it in the sequence selector then right click on it and go to Show atoms gt Sidechain and CA I l 400m in On Argao Notice how the positively charged Arginine is making an electrostatic interaction with the negative phosphate from the DNA backbone 36 HOLE LBU Gyo ARO ASH JER PHE U VAL API VAL CYS ALA CYS PRO GY ARI ASP ARG ARG THA i 1 W 2 e A a aa i aa RFA 2a Aa e IO NA M TBS E Pi i i hiai i r F E r i aa T ea rie fiear faa O R Ta Oty al Samo R273 makes an electrostatic interaction with the DNA phosphate groups Let s see what would happen to the interaction energy between the DNA and P53 when we mutate this Arginine to Alanine Right click on this Arg273 in the sequence selector and go to A number of menus is now presented and here 1s what you need to do in each menu Select Calculate interaction energy change Select Ala Move neighbours and Show disrupted and new hydrogen bonds Don t change any numerical options in the last menu et ea 37 E opie l 4 Ebi of dabii Po opiewa and viaii iie piini i rai aij Lire reger gat Stirn diarii atri rre Prarie bonds jaa She vav clashes yT and madani i a gt a a SS _ ell K IEU LEU GY ARG ASH ER ME HU WA VAL CYS ALA CFS PRO
28. ptions 1 Move neighbours this lonely option is on by default and allows residues around the mutation spot to be moved to accomodate the new residue In case you don t want these neighbours to move uncheck the option Zoom to mutation site checking this option will automatically close up to the mutation site for quick visual inspection Show disrupted and new hydrogen bonds checking this option adds hydrogens to the structure and shows the hydrogen bonds in the wild type and mutated structure Show VdW clashes in WT and mutant checking this option will highlight in red Vander Waals clashes in wild type and mutant structures Tope boar ri Bidii H cis Poh cette ted vice ied eel ch rato sdf von mc sa arte ae ate cea Sa Grp and nem Prroni bones fae Show Vets clashes a WT nd mutant orgs Hyak First options menu Set FoldX options 2 Number of runs This option tells the algorithm how many times it should do the specified mutations Normally it should be set to 1 However sometimes it can be set to higher numbers typically 5 to see if the algorithm has achieved convergence or in other words if the solution offered is the optimal or a trapped solution Each run will result in an additional model for a certain mutation Temperature Temperature K 20 pH At the moment only used for pH effects on metal binding Will not affect the protonation of charged groups in this version onic strengt
29. rst it looks for all Asn Gln and His residues and flips them by 180 degrees This is done to prevent incorrect rotamer assignment in the structure due to the fact that the electron density of Asn and Gln carboxamide groups is almost symmetrical 14 and the correct placement can only be discerned by calculating the interactions with the surrounding atoms The same applies to His 2 We doa small optimization of the side chains to eliminate small VanderWaals clashes This way we will prevent moving side chains in the final step 3 We identify the residues that have very bad energies and we mutate them and their neighbours to themselves exploring different rotamer combinations to find new energy minima We refer to the FoldX manual for an even more detailed explanation on repairing PDB structures Stability of object The FoldX Stability command calculates the free energy of unfolding delta G This 1s the difference in free energy between the folded state and the unfolded state Stability of object calculates the free energy difference of a selected Object To get the most accurate results we recommend to use the Repair object command first It should be noted that stability values in contrast to stability changes do not reflect experimental values but large gt 10kcal mol positive values might indicate a problem in the overall architecture of the structure H HHH cab ce WAT Te a ce Alors The stability in kcal mol i
30. s shown in the YASARA console 15 Alanine scan of object Alanine scan of object mutates every residue in an object to alanine and calculates the energy difference upon mutation to alanine The results of the alanine scan are displayed in the console see screenshot Press the spacebar twice to enlarge the console The same results are also listed in a tab delimited text file called FOLDXSUMMARY out in the cache folder This file can be saved in a custom folder alone or together with all other output files using the option Save last calculation fee Result of an alanine scan is displayed in the console To get the most accurate results we recommend to use the Repair object command first Energy analysis of object Energy analysis of object prints a full energy decomposition per residue in the selected objects aaa ll ll SS Ci This command runs the FoldX SequenceDetail command and prints the energy contribution per residue in the structure as well as a decomposition of the per residue energy in its separate terms Other per residue information such as dihedral angles residue burial and secondary structure is also printed The results are stored in the FOLDXSUMMARY out file which an be saved by choosing Save last calculation To get the most accurate results it is necessary to run the Repair object command first Interaction energy of molecules This option executes the FoldX AnalyseComplex co
31. ues that will not move during a RepairPDB 5 or BuildModel The fixed residues will be colored in yellow see screenshot Do not confuse Fixed residues from the FoldX plugin with fixed residues from the simulation menu from YASARA only in YASARA Dynamics and YASARA Structure These are totally independent i Pia low 55 ir Line ph Fixed residues are colored in yel 13 Free residues In case you want to unfix one or multiple residues that have previously been fixed with the Fix residues command you can use the Free residues command The Free residues command unfixes previously fixed residues These freed residues will be allowed to move during a RepairPDB or BuildModel Repair object This command executes the FoldX RepairPDB command RepairPDB minimizes the energy of a protein structure by rearranging the amino acid sidechains in order to get a better free energy of the protein see screenshot RepairPDB only rearranges sidechains not the backbone Residues that should not be rearranged should be fixed by using the Fix residues command Repair object does an energy minization of the selected Object by running the Foldx RepairPDB command and is required to get accurate I results when subsequently calculating the stability interaction energy or when making mutations with Foldx Result of a RepairPDB run with the repaired structure in green The way RepairPDB operates is the following 1 Fi
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