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USINGAUTOLIGANDWITHAUTODOCKTOOLS: ATUTORIAL...............................................................................1
WrittenbyRodneyM.Harris .............................................................................................................................1 TheScrippsResearchInstitute MolecularGraphicsLaboratory 10550N.TorreyPinesRd. LaJolla,California920371000 USA....................................................................................................................1 20August2009 .............................................................................................................................1 Contents...........................................................................2 Introduction......................................................................4 Before We Start.........................................................................4 FAQ Frequently Asked Questions......................................5 Overview: Using AutoLigand..............................................6 Exercise One: PDB Files Are Not Perfect: Editing a PDB File. .7 Procedure:....................................................................................8 Exercise Two: Preparing the Macromolecule. ....................11 Procedure:..................................................................................11 Exercise Three: Preparing the Grid Parameter File. ...........12 Procedure:..................................................................................12 Exercise Four: Starting AutoGrid 4....................................15 Procedure:..................................................................................15 Exercise Five: Running AutoLigand via Command Line........16 Exercise Six: Running AutoLigand via the GUI...................19 Exercise Seven: Visualizations. .....................................21 Files for Exercises:..........................................................22 Input Files:..................................................................................22 Results Files................................................................................22 Macromolecule.................................................................................................22 AutoGrid..........................................................................................................22 AutoLigand......................................................................................................22
Appendix 1: PMV Basics...................................................23 Modifier........................................................................................................23 Pick..........................................................................................................23 Rotate.......................................................................................................23 Shift..............................................................................................................23 Key...............................................................................................................23 R..........................................................................................................23 Appendix 2: Dashboard Widget.........................................25
Introduction
AutoLigandisatoolusedtoidentifyandcharacterizeligandbinding sites.ThistutorialwillshowyouhowtouseAutoLigand.Wewill
BeforeWeStart
AndonlyifyouareatTheScrippsResearchInstituteThese commandsareforpeopleattendingthetutorialgivenatScripps.We willbestartingthegraphicaluserinterfacetoAutoDockfromthe commandline.Todothis,youneedtoopenaTerminalwindowand thentypethisattheUNIX,MacOSXorLinuxprompt: % source /tsri/python/share/bin/setpath4.csh % cd Desktop/AL_tutorial
FAQFrequentlyAskedQuestions
1. WhereshouldIstartADT? YoushouldalwaysstartADTinthesamedirectoryasthe macromoleculeandmapfiles.YoucanstartADTfromthe commandlineinaTerminalbytypingadtandpressing <Return>or<Enter>. 2. ShouldIalwaysaddhydrogens? Yes.Forthemacromoleculeyoushouldalwaysadd hydrogens,computeGasteigerchargesandthenyoumust mergethenonpolarhydrogens.Polarhydrogensare hydrogensthatarebondedtoelectronegativeatomslike oxygenandnitrogen.Nonpolarhydrogensarehydrogens bondedtocarbonatoms. 3. HowmanyAutoGridgridmapsdoIneed? TorunAutoLigand,youonlyneedC,OAandHDmapsplus theelectrostaticsemapandthedesolvationdmap. 4. HowbigshouldtheAutoGridgridboxbe? Thegridvolumeshouldbelargeenoughtocovertheentire receptorwithafewextraineachdimension.Important: thegridsshouldbedoneata1spacing.Iftheproteinis verylarge,thenyoucanbreakitupintooverlappinggridsand runAutoLigandoneachofthesegridsets,e.g.onecovering thetophalfandonecoveringthelowerhalf.
Overview:UsingAutoLigand
AutoLigandisruntoidentifyand/orcharacterizeligandbindingsites andrequiresa1gridandmapfilesgeneratedbyAutoGrid.Therun
resultisafillvolumeofcontinuouslyconnectedpointsoutputina PDBfileformat.
WhentheAutoLigand GUIisused,asinglefillisgeneratedstarting attheuserspecifiedpoint.Thestartingpointcaneitherbeselectedby clickingonanatomandadjustingthecrosshairswithsliderbars,or enteringcoordinatesinthexyzboxes. WhenAutoLigand isrunfromthecommandline,thecodewillruna singlefillifinputcoordinatesaregivenorscantheentiregridboxand outputthetenbestfillsifnoinputstartingpointisgiven.Byusing differentfillsizesandplottingthetotalenergypervolumeofthefills, optimalbindingsitesonthereceptorcanbeidentified. ItisimportanttonotethatsinceAutoLigandisrunusinga1grid anddockingisnormallyrunusinga0.375grid,itisbesttosetupa subdirectoryfortheAutoLigandmapfilesandresults.Bycopying thesamereceptorpdbfiletothatsubdirectory,allAutoLigandresults canbeviewedinthesamecoordinateframeasdockingresults.
ExerciseOne:PDBFilesAreNotPerfect:Editinga PDBFile
Intheexerciseswhichfollow,weshowyouhowtofindandgenerate fillvolumesforligandbindingsites.TorunAutoLigandrequiresthe followinginputfiles: aPDBQTfileforthereceptor; agridparameterfile(GPF)fortheAutoGridcalculation ProteinDataBank(PDB)filesmayhaveavarietyofproblemsthat needtobecorrectedbeforetheycanbeusedinAutoGrid.These potentialproblemsincludemissingatoms,addedwaters,morethan onemolecule,chainbreaks,alternatelocationsetc. AutoDockTools(ADT)isbuiltonthePythonMoleculeViewer (PMV),andhasanevolvingsetoftoolsdesignedtosolvethesekinds ofproblems.Inparticular,twomodules,editCommandsand repairCommands,containmanyusefultoolsthatpermityoutoadd orremovehydrogens,modifyhistidineprotonation,etc.ADTcannot doeverything,though,anditissometimesnecessarytouseother softwaretocleanupandcheckthequalityofthestructures.Seethe listofresourcesattheAutoDockwebsiteforalistofsomeofthese tools(http://autodock.scripps.edu/resources).
Note:Becauseoftime(andsanity) considerations,inthistutorialweonly demonstrateremovingheteroatomsfromthe receptormolecule.Nevertheless,anyother problemsinthereceptormustbecorrected also.Rememberthatallhydrogensmust alwaysbeaddedpriortousingADTspecific commandstoformatthereceptor.
Note,thatinsomecases,youmaywanttoleaveselectedwaters, substrates,metalions,etc.inthePDBfiletoinfluencethe AutoLigandresults.Inthisexercisewewillstartwithaclean moleculetolookforbindingsites. Inthisexercise,youwillworkonthereceptormacromolecule.You willlearnhowtoremovenonreceptormolecules,howtoaddthe hydrogensthatAutoGridexpects,andhowtosavethemodifiedresult. First,lookatyourPDBfilebystartingADT. [ifrunningthistutorialoutsideofTheScrippsResearchInstitute,you willneedtodownloadthe3B7E.pdbfilefromthePDBwebsite: http://www.rcsb.org/pdb/home/home.do]
Procedure:
1. Readinthereceptormolecule:
Note:Commandsavailableviathe Dashboardcanalsobeinvokedusingthe menusatthetopofthePMVGUI.Here youcouldusethismenusequencetoopen thefilebrowser: File>Read Molecule SeeAppendix2forinformationaboutthe Dashboard.
PositionthecursoroverPMV MoleculesintheDashboard (Appendix2)andclickwiththerightmousebutton.Thiswill openafilebrowser,showingallthefilesinthecurrent directory.Select3B7E.pdbandclickonOpen. ThisloadsaMoleculenamed3B7EintoADT.3B7Eisthe N1receptorinswineflu(moredetailsaboutthismoleculecan befoundonthePDBwebsite).Thebondsbetweenbonded atomsarerepresentedaslineswhilenonbondedatoms,suchas metalionsandoxygenatomsofwatermolecules,areshownas smallsquares.Thenonbondedatomsyouseeherein3B7E aretheoxygenatomsofwatersthatwerepresentinthecrystal structure.Wewillremovethesewaterslater.
2.Color3B7Ebyatomtype InthePMVMoleculesrowoftheDashboard,clickonthe diamondunderAtomtocolorthelinesbyAtomType. Nowthelinesrepresentingtheatomsarecoloredaccordingto thechemicalelement,asfollows: r Carbonsthatarealiphatic(C)white, q Carbonsthatarearomatic(A)green, q Nitrogens(N)blue, q Oxygens(O)red, q Sulfurs(S)yellow, q Hydrogens(H)cyan.
ClickAll Geometries andthenclickOK.Thenhitthe deselectionbutton(pencileraserbutton). 6. TopreparethismoleculeforAutoLigandruns,wewillexit ADTandeditthePDBfiledirectly.Ascanbeseen,thefile containstwochainsofadimer,eachwithaligand,andwater andmetalionsatvariouslocations.Theeasiestwaytoclean upthisfileistoselectonechainandstripawayallotheratoms notinthischain.[Notethatthemoleculecanbecleanedupin ADT,butwhentherearemultipleheteroatomsotherthanjust water,itiseasiertoeditthePDBfiledirectly.] ExitADTclicktheXinupperright. 7. Copy3B7E.pdbtonewfiletoedit: cp 3B7E.pdb 3b7e.pdb [Note:itisjustmyconvention touseuppercaselettersforthefullPDBfileandlowercasefor themodifiedPDBfileyoucanuseanynamingyoulike] 8. Editfilewithvieditor(oryourfavoriteeditor): vi 3b7e.pdb SearchforchainB(line3694inthisfile).Deletealllines abovethisone: :1,3693d(note,commandsmaybedifferentinyoureditor) FindtheendofchainB: /TER Notethelinenumber(2981inthiscase).Gototheendofthe file(line4282inthiscase)anddeleteeverythingbelowchain B:
ExerciseTwo:PreparingtheMacromolecule.
Procedure:
1.File Read molecule
CAUTION:TheAutoGridcalculationmust bebasedontherigidresiduesonly.
Select3b7e.pdb(notethelowercaselettersisthefilewe modifiedfromexerciseone)Click:Open 2.Edit Hydrogens Add Click:OKtoacceptdefaults. 3.Grid Macromolecule Choose... Select3b7e Selectingthemacromoleculeinthiswaycausesthefollowing sequenceofinitializationstepstobecarriedoutautomatically: ADTchecksthatthemoleculehascharges.Ifnot,itadds Gasteigerchargestoeachatom.Rememberthatall hydrogensmustbeaddedtothemacromoleculebeforeitis chosen.Ifthemoleculealreadyhadcharges,ADTwould askifyouwanttopreservetheinputchargesinsteadof addingGasteigercharges. ADTmergesnonpolarhydrogensunlesstheuser preferenceadt_automergeNPHSissetnottodoso. ADTalsodeterminesthetypesofatomsinthe macromolecule.AutoDock4canaccommodateany numberofatomtypesinthemacromolecule.
ClickOKontheWARNINGdialogbox.Theformshouldbepre filledinas3b7e.pdbqt.ThenclickonSave.
ExerciseThree:PreparingtheGridParameterFile.
Procedure:
1.Grid Set Map Types Directly... EdittheselectionsintheAutoGpfLigandwidgettoinclude onlyC,HD,andOA. ClosethiswidgetwiththeAcceptbutton.
2.Grid Grid Box OpenstheGridOptionsWidget.Firstabrieftourofthis widget: Thishasmenubuttonsatthetop:File,Center,Viewand Help. File ThismenuletsyouclosetheGridOptionsWidget, whichalsocausesthegridboxtodisappear.Youcan Close saving current valuestokeepyourchanges orClose w/out savingtoforgetyourchanges. Center Thismenuletsyousetthecenterofthegridboxinfour ways: Pick an atom, Center on ligand, 1
Center on macromoleculeor On a named atom. View Thismenuallowsyoutochangethevisibilityofthe boxusingShow box,andwhetheritisdisplayedas linesorfaces,usingShow box as lines.Thismenu alsoallowsyoutoshoworhidethecentermarkerusing Show center markerandtoadjustitssizeusing Adjust marker size. TheGridOptionsWidgetdisplaystheCurrentTotalGrid Pointspermap.Thistellsyouhowbigeachgridmapwill be:(nx+1)x(ny+1)x(nz+1),wherenxisthenumberof gridpointsinthexdimension,etc. Thishas3thumbwheelwidgetswhichletyouchangethe numberofpointsinthex,yandzdimensions.Thedefault settingsare40,40,40,whichmakesthetotalnumberof gridpointspermap68921becauseAutoGridalwaysadds oneineachdimension. Youwillalsonoticeithasathumbwheelthatletsyou adjustthespacingbetweenthegridpoints. Therearealsoentriesandthumbwheelsthatletyouchange thelocationofthecenterofthegrid.
Thenumberofpointsineachdimensioncanbeadjustedupto126. AutoGridrequiresthattheinputnumberofgridpointsbeaneven number.Itthenactuallyaddsonepointineachdimension,since AutoGridandAutoDockneedacentralgridpoint. Thespacingbetweengridpointscanbeadjustedwithanother thumbwheel.Thedefaultvalueis0.375betweengridpoints,which isaboutaquarterofthelengthofacarboncarbonsinglebond.Grid spacingvaluesofupto1.0canbeusedwhenalargevolumeistobe investigated.Ifyouweretoneedgridspacingvalueslargerthanthis, youcouldedittheGPFinatexteditorbeforerunningAutoGrid. Forthisexercise: Thegoalistogenerateagridboxthatencompassestheentirereceptor moleculeata1spacing.First,adjustthespacingthumbwheelto
ExerciseFour:StartingAutoGrid4
Ingeneral,youshouldknow:
AutoGrid 4(andAutoLigand)mustberuninthe
Procedure:
1.Run Run AutoGrid IntheRunAutoGridwidgetthatopens,changeautogrid3to autogrid4andpressthe<Return>key. 2.Launch StartstheAutoGrid4job.Onmostplatforms,thisopensa ProcessManagerthatallowsyoutoseespecificsaboutcurrent AutoGridandAutoDockjobs.Itisalimitedprocessmanager thatyoucanusetoterminateanAutoGridorAutoDock processbyselectingitsentry.Youareaskedifyoureallywant tokillit. (TheAutoGrid4calculationtakesabout2minutesonaMac.) Pleasenotethatyoucaneasilystartajobfromthecommandline: % autogrid4 p 3b7e.gpf l 3b7e.glg & 1
ExerciseFive:RunningAutoLigandviaCommand Line.
Forthisexercise,wewillopenanewterminalwindowandchangeto thedirectorywherethemapfilesarelocated.TorunAutoLigandand findthetenbestbindingsites,typethefollowing: python [PATH]AutoLigand.py 3b7e 90 wherethe3b7eisthebasenameofthemapfilesand90isthenumber offillpointstouse(forthisexercise).[Note,youmayneedtoadjust yourPATHtotheAutoLigand.pycommanddependingonhowyou havesetupyourcomputer.] Thecommandtakes10ormoreminutestorundependingonthe numberoffillpointsyouhaverequested(500pointscantakehours). ThecodegeneratesuptotenFILLfilesandoneResultsfile.Someof thetoptenfillresultsmaycollapsetothesameanswer,thusreducing thenumberoffillstolessthanten.Theresultsarelistedinrankorder frombestTotalEnergyperVolumeofeachofthefillsgenerated. Thisexerciseshouldgenerateninefillsfortheinputsizeof90points. EachfillfileisoutputasaPDBfileinthefollowingformat: FILL_90out1.pdb where90isthenumberofpointsinthefillandout#istherankofthat fill.Toexaminethefillvolumes,gobacktotheADTwindowanddo thefollowing: 1.File Read Molecule FILL_90out1.pdb ThisopensthefirstfillvolumefoundbyAutoLigand.The defaultinADTistoopeninLinemode.Tomakeitmore visible,wewillchangeittoamodifiedCPKmode. LeftclickonthecircleunderCPKintheFILL_90out1lineon theDashboard.Then,RightClickthiscircletoopenasize widgit.MovetheScaleFactorThumbwheelto0.3andclick OK.Lastly,clickonthediamondunderAtominthe 1
Fills3and6sharesomeoverlap,butaremuchdifferentfills. Fills4and5arealmostidentical.Andfills7and8sharesome overlap. Fills1,2and4,5areinternalwithalotofHbonddonorsand acceptors,whichisanindicationthattheymightbeinternal structuralwatersites.Tochecktoseeiftheyarestructural watersites,wewillopentheoriginalPDBfileandhighlight thewaters. 4. File Read Molecule 3B7E.pdb NoteuppercaseletterswhichistheunmodifiedPDBfile. Next,wewillselectthewatersandhighlightthem. 5.Select Select From String Type:HOH*intheResidueboxandthenclickAddandthen Dismissthewidgit.Allofthewatershavebeenselectedand shouldhaveyellowcrossesonthem. Next,wewilldisplaythewatersinCPK. 6. Display CPK ChangetheScaleFactoronthewidgit0.5sothattheywill standoutfromthefillvolumes.Then,shutofftheselection (thepencilerrasericonbutton). Focustheviewonfills1and2.Itcanbeseenthatthefills overlap9watermolecules.Thegreysquareonthedashboard
canbeusedtohide/showthefills,whichisusefulin determiningoverlaps. Fills3and6overlaptheZanamivirligandmoleculefromthe crystalstructure. Now,focustheviewonfills4and5.Thisinternalsite contains8watermolecules.Thus,thetwointernalsitesarenot likelytobeligandbindingsites. Fills7and8overlapaboundGlycerolinthecrystalstructure. Fill9,whichhasthelowesttotalenergypervolume,lookslike asurfacepatchandmaynotbearealligandbindingsite. Toshutoffthewatermolucules,clickontheCPKbuttonon the3B7Eline,andclickitagaintoshutitoff.
ExerciseSix:RunningAutoLigandviatheGUI.
FromexerciseFive,wefoundtheligandbindingsite.Inthis exercisewewillusetheGraphicalUserInterface(GUI)torun morefillsstartingonlyattheligandbindingsite. RestartADTandopen3b7e.pdbqt. 1. File Read Molecule 3b7e.pdbqt Selectcolorbyatomtypesbyclickingonthediamondunder Atominthe3b7erow. Next,wewillopenthezanamivirligandPDBfile.[Note,to nonTSRIclassusers:thisfilecanbegeneratedfromthe originalPDBfilebydeletingalllinesbuttheZMRBhetero atomsHETATM 6021toHETATM 6043andsavingas zanamivir.pdb] 2. File Read Molecule zanamivir.pdb Highlightthisligandbychangingittoballandstick.Clickthe circleunderS&Binthezanamivirrowandthencolorbyatom typebyclickingonthediamondunderAtominthezanamivir row. Now,wewillbeginmakingdifferentsizeAutoLigandfillsat thissite. 3. Compute AutoLigand Run AutoLigand... Thisopensthewidgitandshowsaboxaroundthespacewhich canbesearched.TheFileBaseNameisprefilledwiththe nameofthemapfiles(3b7einthiscase)andsetto100points. Thestartlocationisdefaultedtothecenterofthegridboxor thelaststartpointused.Therearethreewaystochangethe startingpoint: 1. Clickonanatomnexttowhereyouwishtostartfilling. 2. EntertheXYZcoordinates 3. Adjustthesliderbars Wewanttostartmakingafewdifferentsizefillsstartingata pointnexttotheligand. 1
Clickontheoxygenatominthecenterringoftheligand(any pointnearthiswouldworkthesame,butthisatomiseasytofind). Thecoordinatesandsliderbarsautomaticallyadjust.Note,thatthe ligandisnotreallythereforAutoLigandcalculationsasthemapfiles weregeneratedwithouttheligandinplace.ClickOKtostartthe calculation.[ImportantNote:ifotherfillsarerunatthesamesize, thefillswillbeoverwritten.Usedifferentnumberofpoints,orrename theFILLfileifyouwishtoruntheGUIagainandkeepyourfirst results.] Now,runafewdifferentsizefills. 4.Compute AutoLigand Run AutoLigand... ChangetheNumberofPointsto150.Note,thatthestartpoint islocatedatthesamepositionasthelastrun(aslongasyou arestillinthesameADTsession).ClickOKtostartthe calculation. Rerunagainforsizes200and250. Toseehowthefillsfitinthepocket,wewillmakeamolecular surfaceaboutthereceptormolecule.Clickthecircleunderthe MScolumninthe3b7erow. Youcanseeaslargerfillsizesareused,thefillvolumeis forcedtopushoutofthebindingpocketandmoveintoregions oflessaffinity.AResultsfileisgeneratedforeachrunofthe GUI(noteagainthatifthesamefillsizeisused,thesefilesare overwritten).Bylookingattheenergypervolume(EPV)of fills100to250,youcanseethattheEPVstaysaboutthesame forfills100and150,butdropsoffafterthat.
ExerciseSeven:Visualizations.
by Properties
FilesforExercises:
InputFiles:
3B7E.pdb
ResultsFiles
Macromolecule
3b7e.pdbqt
AutoGrid
3b7e.gpf 3b7e.glg 3b7e.*.map 3b7e.maps.fld,3b7e.maps.xyz
AutoLigand
3b7e_*Results.txt FILL_*out*.pdb
Appendix1:PMVBasics
None
Pick
Rotate
Translateleft/right(X) andup/down(Y)
Shift
Select
Scaleor Zoom
Translatein/out(Z)
Youcanalsopressthefollowingkeysintheviewerwindowto changetheviewofthemolecule:
Appendix2:DashboardWidget
Selectentry+CommandButtons TreeWidget
Note:Clickingonashaperectangle,circle, squareordiamondunderacommandcauses thecommandlinkedtotheshapetobeapplied toeachnodeinthecorrespondingrow.Ifthe shapeisoff(coloredwhite),thecommandwill beappliedtonodesandtheshapewillbe coloredred.Iftheshapeison(coloredred), clickingonthecommandbuttonwillundothe commandandtheshapewillbecoloredwhite. Circlesareusedfordisplaycommands,squares forlabelcommandsanddiamondsforcolor commands.Coloringcanbereplacedbya differentcoloringschemebutcannotbeundone. Thegrayrectangleisusedforshow/hideand thewhiterectangleforselect.
TheTreeWidgetontheleftlistsallmoleculescurrentlyloadedin PMV.Clickonthearrows tonavigatebetweenmolecules , chains ,residues andatoms .Clickingonashapein oneofthecolumnsintherightsectionexecutesthePMV commandcorrespondingtothelabelatthetopofthecolumnon thegroupofnodescorrespondingtotherow.There16different commandsthatcanbeexecutedthiswaygrayrectangle (Show/Hide),select/unselect(Sel.),displaylines(Lines),display CPK(CPK),displaysticksandballs(S&B),displaysecondary structure(Rib.),displaymolecularsurface(MS),displaylabels (Lab.),colorbyatomtype(Atom),colorbymolecule(Mol),color bychain(Chain),colorbyresidueaccordingRasmol(RAS),color byresidueaccordingShapely(SHA),coloraccordingtoDavid Goodsellcolors(DG),colorbysecondarystructureelementtype (Sec.Str.)andcolorbyinstance(Inst). Tohelpusersseetheconnectionbetweenmolecularfragmentsand PMVcommands,acrosshairisdrawnwhencursorisinsidethe Dashboardwidget. Rightclickingonashapedisplaysaninputparameterpanelforthe commandandallowstheusertocustomizespecificinput parametersforthecommand.
aselectednode,thecorrespondingcommandisappliedtoall currentlyselectednodes. Hoveringoverthisentryshowssamples oftherequiredsyntax. Theoptionmenuonthetopallowstheusertospecifywhether commandsshouldbeappliedtothebackboneatomsonly(BB),the sidechainatomsonly(SC),thesidechainatomsandCAatoms (SC+CA)orthefullmolecularfragment(ALL).Thissettingcan beoverriddenforeachcolumn(CMD). ClickonthegrayrectangleunderShow/Hide.Noticethatthe moleculeintheviewerdisappears.Clickonthesamerectangle againtoredisplayit.ClickontherectangleundertheSellevelto selectordeselectall.Experimentbyclickingoneachoftheother buttons.Theseareshortcutstoabasicsetofcommandsfor displayingandcoloringvariousmolecularrepresentations.