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UsingAutoLigandwith AutoDockTools: ATutorial

Written by Rodney M. Harris


The Scripps Research Institute Molecular Graphics Laboratory 10550 N. Torrey Pines Rd. La Jolla, California 92037-1000 USA 20 August 2009

Contents

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

usebothacommandlineinterfaceandaGraphicalUserInterface calledAutoDockTools,orADT,tosetupandrunAutoLigand jobs. PreviousexperiencewithADT andAutoGridisrecommended.

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.

Note:EachrowintheDashboardislinkedtoa singleentity.TheentityiseitherPMV Moleculeswhichisthegroupcomprisedofall theMoleculesintheViewer,asingle Molecule,asingleChain,asingleResidueor asingleAtom.Thediamondsontheright sideoftheDashboardareusedtocolorthe displayedgeometriesforthatrowsentityby differentcoloringschemes.

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.

3.ColorbyChain Inthe3B7ErowoftheDashboard,clickonthediamondunder Chain.Thiscolorsthetwochainsblueandgreen.To highlighttheboundzanamivir(theRelenzadrug)molecules, dothefollowing:


Select Select From String

TypeZMR*intheResidueentry,thenclickAdd.Click DismisstoclosetheSelect From Stringwidget.Withthe residuesselected,changethemtoballandsticktohighlight:


Display

Sticks and Balls

Click OK. Thencolorbyatomtype:


Color by Atom Type

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:

:2981,4282d Savethefile::wq Wewillusethisnewfile,3b7e.pdb,tosetupgridsforAutoGrid.

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.

Note:IfADTmakesanymodificationstothe moleculeyouopened,afilebrowserwillopen foryoutospecifyafilename.Typeinaname+ .pdbqtandclickonSave.

ClickOKontheWARNINGdialogbox.Theformshouldbepre filledinas3b7e.pdbqt.ThenclickonSave.

ExerciseThree:PreparingtheGridParameterFile.

ThegridparameterfiletellsAutoGrid4whichreceptortocompute thepotentialsaround,thetypesofmapstocomputeandthelocation andextentofthosemaps.Inadditionitmayspecifyacustomlibrary ofpairwisepotentialenergyparameters.ForAutoLigandonlytheC, OA,andHDmapsareusedplusanelectrostaticsmapandaseparate desolvationmap.

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.

Note:clickingwiththerightmouse buttononathumbwheelwidget opensaboxthatallowsyoutotype inthedesiredvaluedirectly.Like manyotherentryfieldsinADT,this updatesonlywhenyoupress <Enter>.

Thenumberofpointsineachdimensioncanbeadjustedupto126. AutoGridrequiresthattheinputnumberofgridpointsbeaneven number.Itthenactuallyaddsonepointineachdimension,since AutoGridandAutoDockneedacentralgridpoint. Thespacingbetweengridpointscanbeadjustedwithanother thumbwheel.Thedefaultvalueis0.375betweengridpoints,which isaboutaquarterofthelengthofacarboncarbonsinglebond.Grid spacingvaluesofupto1.0canbeusedwhenalargevolumeistobe investigated.Ifyouweretoneedgridspacingvalueslargerthanthis, youcouldedittheGPFinatexteditorbeforerunningAutoGrid. Forthisexercise: Thegoalistogenerateagridboxthatencompassestheentirereceptor moleculeata1spacing.First,adjustthespacingthumbwheelto

1.Next,adjustthenumberofpointsofthexyzwheelstocoverthe entiremolecule:60,60,60shouldbeenoughinthiscase.Note,that theboxcouldbeadjustedtoexcludepartsofthemolecule(suchas proteinproteininterfaces)whereyoudonotwantAutoLigandto search. ClosethiswidgetbyclickingFile Close saving current.

3.Grid Output Save GPF Opensafilebrowserallowingtheusertospecifythenameof thegridparameterfile.Theconventionistouse.gpfasthe extension. WritetheGPFas3b7e.gpf Besuretoputintheproperpath

ExerciseFour:StartingAutoGrid4

Ingeneral,youshouldknow:
AutoGrid 4(andAutoLigand)mustberuninthe

directorywherethemacromoleculeandparameterfilesare tobefound. ThenamedfilesintheGPFmustnotincludepathnames. Currently,itisnotpossibletoruneitherprogramona WINDOWSplatformexceptinaCygwinshell.See http://autodock.scripps.edu/faqshelp/faq/howcanilearn moreabouthowtouselinuxandcygwinand http://autodock.scripps.edu/faqshelp/faq/installing autodockonwindowsformoreinformationaboutCygwin.

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

2.File Read Molecule FILL_90out2.pdb Thisfilloverlapsmostoffill1.Nearlyidenticalfillscanbe generatedwhenthefillsarestartedfromdifferentpointsatopposite ends. 3. Opentherestofthefills:


File Read Molecule FILL_90outX.pdb

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.

Tovisualizetheresults,amolecularsurfacecanbeplacedaroundthe fillvolumes.ClickthecircleundertheMScolumninthe FILL_150out1row.ThenclickcolorbyAtomtype.TheDejaVuGUI buttoncanbeusedtomodifythefillresultasneeded. Oneotherimportantvisualizationthatcanbeseenistocolorby affinityvaluesperpointinthefill.Thisdemonstratesthatligand bindingsitesconsistofhotpointsconnectedbylesseraffinity structure. SelectFILL_150out1(usetheselectsquareonthedashboard).


Color

by Properties

ClickcpkandOK,thenclicktheAtombuttonandusescrollbarto movedowntobottomoflistandselecttemperatureFactor.Next, clickthe>>buttoninthecenteratthebottomofthewidgit,thenclick OK.Thisaffinityscaleisfromred(best)toblue(worst)andshows threeregionsofhigheraffinityconnectedbyloweraffinitystructurein thisexample.

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

Youmighthaveathreebuttonmouse.Ifso,themousebuttonscanbe usedaloneorwithamodifierkeytoperformdifferentoperations.To zoomthemolecule(makethemoleculelookbiggerorsmaller)inthe viewerwindow,pressandholddownthe<Shift>keyandthenclickand dragwiththemiddlemousebutton.Tosummarizewhatthemousebuttons do:


Modifie r Left Middle Right

OnAppleComputers:. option:Rotate command:Translateleft/right shift+option:ScaleorZoom shift+command:Translatein/out

None

Pick

Rotate

Translateleft/right(X) andup/down(Y)

Shift

Select

Scaleor Zoom

Translatein/out(Z)

Note:asyoutranslateamoleculeoutintheZ dimension,itwilldisappearintofogwhichisused fordepthcueing.

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.

Note:AselectionintheTreeisusedtobuilda groupofnodestobethetargetforcommands linkedtoshapes.Itisnotthesameasthecurrent selectionintheViewer.Itcanbeselectedusing theappropriaterectangles.

TheSel:entryinthetopleftcorneroftheDashboardcanbeused toselectentriesintheTreeusingaPmvcompoundselector.Nodes matchingthespecifiedstringwillbeselected.Selectednodesare outlinedwithayellowselectionbox.Whenashapeisclickedfor

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.