1. GROMACSTutorial GROMACSIntroductoryTutorial Gromacsver3.3.1 Author: JohnE.Kerrigan,Ph.D. AST/IST,UniversityofMedicineandDentistryofNJ 675HoesLane Piscataway,NJ08554 Phone: (732)2354473 Fax: (732)2355252 Email: kerrigje@umdnj.edu 1

2. GROMACSTutorial GROMACSTutorialforSolvationStudyofSpiderToxinPeptide. Yu,H.,Rosen,M.K.,Saccomano,N.A.,Phillips,D.,Volkmann,R.A.,Schreiber,S.L.: SequentialassignmentandstructuredeterminationofspidertoxinomegaAgaIVB. Biochemistry32pp. 13123(1993) GROMACSisahighend,highperformanceresearchtooldesignedforthestudyofprotein dynamicsusingclassicalmoleculardynamicstheory.[1] ThisversatilesoftwarepackageisGnu publiclicensefreesoftware.Youmaydownloaditfrom http://www.gromacs.org .GROMACS runsonlinux,unix,andonWindows(arecentdevelopment). Synopsis.Inthistutorial,youwillstudyatoxinisolatedfromthevenomofthefunnelwebspider. Venomtoxinshavebeenusedinthepasttoidentifycationchannels.Calciumionchannelsregulate theentryofthisionintocells.Nervesignalsarehighlygovernedbythebalanceofionsinneuronal cells.Itishypothesizedthatexposedpositivelychargedresiduesinvenomslikethespidertoxin herebindfavorablytothenegativelychargedentranceofthecellsionchannel.Thespidertoxinin thistutorialhasitspositivelychargedresiduespointingpredominantlytoonesideofthepeptide. Theionchannelisblocked,resultinginblockednervesignalleadingtoparalysisandultimatelyto death(presumablyviarespiratoryfailure). Wewillstudythispeptidetoxinusingexplicitsolvationdynamics.First,wewillcompareanin vacuomodeltoasolvatedmodel.Wewillsolvatethepeptideinawaterboxfollowedby equilibrationusingNewtonslawsofmotion.Wewillcompareandcontrasttheimpactof counterionsintheexplicitsolvationsimulation.Wewillseekanswerstothefollowingquestions: Isthesecondarystructurestabletothedynamicsconditions? Arethesidechainsofthepositivelychargedresiduespredominantlydisplacedtoonesideofthe peptidestructure?Dothecounterionsholdthesepositivelychargedresiduesin placeordothey movearound? Whatroledoeswaterplayinmaintainingthestructureofproteins? Note:Youwillgenerategromacs(*.gro)structurefilesinthistutorial.Toviewthesefiles,you mustuseVMD(Downloadfrom:http://www.ks.uiuc.edu/Research/vmd/). Inaddition,you shouldobtainacopyoftheGROMACSusermanualat http://www.gromacs.org . Download1OMB.PDBfromtheProteinDataBank(http://www.rcsb.org/pdb/). ItisadvisabletouseDeepView(DownloadDeepViewfrom http://www.expasy.ch/spdbv/) to previewthefileifyouknowthatyourstructuremaybedisordered(i.e.residueswithmissingside chains).DeepViewwillreplaceanymissingsidechains(However,bewareasDeepViewwillmark thoserebuiltsidechainswithastrangecontrolcharacterthatcanonlyberemovedmanuallyusinga 2

3. GROMACSTutorial texteditor!). Therearenomissingsidechainsinthispdbfile,sowewillnotworryaboutthatin thisexercise. Createasubdirectoryinyourunixaccountcalledfwspider.Withinthisnewdirectory,createthe followingdirectories:invacuo,wet,andionwet.Usesftptocopythe1OMB.pdbfiletothe fwspidersubdirectories(placeacopyineachsubdirectorywithinthefwspiderdirectory). (IMPORTANT!WheneveryousftpatextfiletoaunixsystemfromWindows,besuretoconvert thefiletoaunixtextfile.Dothisusingtheto_unix command(e.g. to_unix filenamefilename convertsfilename toaunixtextfile. InRedHatLinux,usethedos2unix command. Texteditorsin WindowslikeMSWordaddcontrolcharactersthatmaycauseerrorsinunixprograms.) Processthepdbfilewith pdb2gmx.Thepdb2gmx (toviewthecommandlineoptionsforthis commandjusttypepdb2gmx hInfact,togethelpforanycommandinGromacsjustusetheh flag)commandconvertsyourpdbfiletoagromacsfileandwritesthetopologyforyou.Thisfileis derivedfromanNMRstructurewhichcontainshydrogenatoms.Therefore,weusetheignhflag toignorehydrogenatomsinthepdbfile.Theffflagisusedtoselecttheforcefield(G43a1isthe Gromos96FF,aunitedatomFF).Thef flagreadsthepdbfile.Theoflagoutputsanewpdb file(variousfileformatssupported) withthenameyouhavegivenitinthecommandline.Thep flagisusedforoutputandnamingofthetopologyfile.Thetopologyfileisveryimportantasit containsalloftheforcefieldparameters(basedupontheforcefieldthatyouselectintheinitial prompt)foryourmodel. RecentstudieshaverevealedthattheSPC/Ewatermodel [2]performs bestinwaterboxsimulations.[3] Usethespcewatermodelasitisbetterforusewiththelong rangeelectrostaticsmethods.So,weusethewaterflagtospecifythismodel. pdb2gmx ignhff G43a1f 1OMB.pdb ofws.pdb pfws.topwater spce SetuptheBoxforyoursimulations editconf btcubicffws.pdb ofws.pdb d0.9 Whatyouhavedoneinthiscommandisspecifythatyouwanttouseasimplecubicbox. Thed 0.9flagsetsthedimensionsoftheboxbasedupon settingtheboxedgeapprox0.9nm(i.e.9.0 angstroms)fromthemolecule(s)periphery.Ideallyyoushouldsetdatnolessthan0.85nmfor mostsystems.[4] [SpecialNote: editconf mayalsobeusedtoconvertgromacsfiles(*.gro)topdbfiles(*.pdb)and viceversa.Forexample:editconf f file.groo file.pdbconverts file.gro tothepdbfilefile.pdb] Youmayusethefilesgeneratedfromthethissteptobeginyourinvacuosimulation.Forthein vacuoworkjustmoveaheadtotheenergyminimizationstepfollowedbythemoleculardynamics step(Nopositionrestraineddynamicsnecessaryforinvacuowork.Why?). SolvatetheBox genbox cpfws.pdb csspc216.groofws_b4em.pdb pfws.top 3

4. GROMACSTutorial Thegenbox commandgeneratesthewaterboxbaseduponthedimensions/boxtypethatyouhad specifiedusingeditconf.Inthecommandabove,wespecifythespcwaterbox. Thegenbox programwilladdthecorrectnumberofwatermoleculesneededtosolvateyourboxofgiven dimensions. Setuptheenergyminimization. Usetheem.mdpfile.Gromacsusesspecial*.mdpfilestosetup theparametersforeverytypeof calculationthatitperforms.Lookintothecontentsofthisfile.Itspecifiesasteepestdescents minimizationtoremovebadvanderWaalscontacts.Editthefileandchangenstepsto400.Ifthe minimizationfailstoconverge,resubmitwithnsteps=500.(Theminimizationshouldconvergein lessthan400stepshowever,differentplatformsresponddifferently.)Toresubmitthejob,you willneedtorerungrompp.(Note:thepathtothecpreprocessormaybedifferentonyour machine.Usethewhichcommandtolocate[i.e.whichcpp]!) Contentofem.mdp: title=FWS cpp= /usr/bin/cppthecpreprocessor define= DFLEXIBLE constraints=none integrator =steep dt=0.002ps! nsteps=400 nstlist=10 ns_type=grid rlist=1.0 coulombtype=PME rcoulomb=1.0 vdwtype=cutoff rvdw =1.4 fourierspacing =0.12 fourier_nx =0 fourier_ny =0 fourier_nz =0 pme_order =4 ewald_rtol =1e5 optimize_fft =yes Energyminimizingstuff emtol=1000.0 emstep=0.01 Importantaspectsoftheem.mdpfile: titleThetitlecanbeanygiventextdescription(limit64characterskeepitshortandsimple!) cpplocationofthepreprocessor definedefinestopasstothepreprocessor. DFLEXIBLEwill tellgrompptoincludetheflexible SPCwatermodel insteadof therigidSPCintoyourtopology.Thisallowssteepestdescentsto minimizefurther. 4

5. GROMACSTutorial constraintssetsanyconstraintsusedinthemodel. integrator steeptellsgromppthatthisrunisasteepestdescentsminimization.Usecgfor conjugategradient. dt notnecessaryforminimization.Onlyneededfordynamicsintegrators(likemd). nstepsInminimizationruns,thisisjustthemaximumnumberofiterations. nstlistfrequencytoupdatetheneighborlist.nstlist=10(updates thelistevery10steps). rlistcutoffdistanceforshortrangeneighborlist. coulombtype tellsgromacshowtomodelelectrostatics.PMEisparticlemeshewaldmethod (pleaseseetheGromacsusermanualformoreinformation). rcoulombdistanceforthecoulombcutoff vdwtype tellsGromacshowtotreatvanderWaalsinteractions(cutoff,Shift,etc.) rvdwdistancefortheLJorBuckinghampotentialcutoff EMStuff emtol theminimizationconvergeswhenthemaxforceissmallerthanthisvalue(inunitsofkJ 1 1 mol nm ) emstepinitialstepsize(innm). Nowprocessthefileswith grompp. gromppisthepreprocessorprogram(thegromacspre processorgromppGetit!Sigh!). gromppwillsetupyourrunforinputintomdrun. gromppf em.mdpcfws_b4em.pdb pfws.top ofws_em.tpr Thefflagingromppisusedtoinputtheparameterfile(*.mdp).Thecflagisusedtoinputthe coordinatefile(thepdbfile,*.pdb) pinputsthetopologyandooutputstheinputfile(*.tpr) neededformdrun. Usinggenionandthetprfiletoaddions.Youmayusethetprfilegeneratedheretoadd counterionstoyourmodeltoneutralizeanynetcharge.Ourmodelhasanetchargeof+2.00. Therefore,wewanttoaddtwochlorideions.Copythefws_em.tprfilethatyouusedforyour explicitsolvatedmodeltoyourionwetsubdirectory.Inaddition,copyyourfws.topandposre.itp filesfromyourexplicitsolvationmodeltoyourionwetsubdirectory.Usethegenioncommandto addthechlorideions. genionsfws_em.tpro fws_ion.pdb nnameCLnn 2g fws_ion.log Wherennameisthenegativeionname(CLfortheGromosG43a1forcefieldseetheions.itp fileforspecificswrtforcefield),nnisthenumberofnegativeionstoadd.Thegflaggivesa nametotheoutputlogforgenion. Whenyouprocessthiscommand,youwillbepromptedtoprovideacontinuousgroupofsolvent molecules,whichshouldbeGroup12(SOL).Type12then.Youwillnoticethattwo solventmoleculeshavebeenreplacedbyClions.Nowyoumustmakethefollowing additions/correctionstoyourfws.topfile: Add 5

6. GROMACSTutorial #includeions.itp (Note:Thisshouldbepresentbydefaultalreadyinversion3.2&greater) aftertheincludestatementfortheforcefield.Subtract2fromthetotalnumberofSOLmolecules andaddalinefor2Clmoleculesinthe[molecules]sectionattheendofthefile.Youwillalso needtomodifythetemperaturecouplingschemeforyourpr_md.mdpandmd.mdpfiles. Now,youwillusefws_ion.pdbinplaceoffws_b4em.pdbforgeneratingtheactualinputtpr using gromppforenergyminimization. Temperaturecouplingschemeforthepr_md.mdpandmd.mdpfilesafteraddingchlorideions. Berendsentemperaturecouplingison Tcoupl=berendsen tau_t=0.1 0.1 tc_grps =protein nonprotein ref_t=300 300 Remember:Ifyouaddedthechlorideions,youwillneedtorunthegromppstepagain. First removetheoldfws_em.tprfile,thenrunthenextgromppcommandbelow. Weaddedchlorideions inourmodeltoneutralizedtheoverallnetchargeofthemodel. gromppf em.mdpcfws_ion.pdb pfws.top o fws_em.tpr Runtheenergyminimizationinbackground(Usetheampersand&attheendofthecommand). mdrunvdeffnm em (deffnm tellsgromacsthedefaultfilenametousefortherun.Thisisaveryusefulshortcut.) Usethetail commandtocheckontheprogressoftheminimization. tail 15em.log Whentheminimizationiscomplete,youshouldseethefollowingsummaryinyourlogfile indicatingthatsteepestdescentsconverged.Usetail 50em.log SteepestDescentsconvergedtoFmax0.001ps). [7] Studythemdp filebelow. Contentofpr.mdp. title=FWS warnings=10 cpp=/usr/bin/cpp define= DPOSRES constraints=allbonds integrator =md dt=0.002 ps! nsteps=10000 total20.0ps. nstcomm=1 nstxout=250 collectdataevery0.5ps nstvout=1000 nstfout=0 nstlog=10 nstenergy=10 nstlist=10 ns_type=grid rlist=1.0 coulombtype =PME rcoulomb=1.0 vdwtype=cutoff rvdw=1.4 fourierspacing =0.12 fourier_nx =0 fourier_ny =0 fourier_nz =0 pme_order =4 ewald_rtol =1e5 optimize_fft =yes Berendsentemperaturecouplingison Tcoupl=berendsen tau_t=0.1 0.1 tcgrps =proteinnonprotein ref_t=300 300 Pressurecouplingison Pcoupl=parrinellorahman tau_p=0.5 compressibility=4.5e5 ref_p=1.0 Generatevelocitesisonat300K. gen_vel=yes gen_temp=300.0 gen_seed =173529 7

8. GROMACSTutorial Importantthingstoknowaboutthemdpfile. TheDPOSREinthedefinestatementtellsGromacstoperformpositionrestraineddynamics. Theconstraintsstatementisaspreviouslydiscussed. allbondssetstheLINCSconstraintforall bonds.[7] Theintegrator tellsgromacswhattypeofdynamicsalgorithmtouse(anotheroptionissdfor stochasticdynamics). dtisthetimestep(wehaveselected2fshowever,thismustbeenteredinunitsofps!). nstepsisthenumberofstepstorun(justmultiplynstepsXdttocomputethelengthofthe simulation). nstxouttellsgromacshowoftentocollectasnapshotforthetrajectory.(e.g.nstxout=250with dt=0.002wouldcollectasnapshotevery0.5ps) coulombtypeselectsthemannerinwhichGromacscomputeselectrostaticinteractionsbetween atoms.(PMEisparticlemeshewaldthereareotheroptionslikecutoff). rcoulombandrvdwarethecutoffs(innm1.0nm=10.0angstroms)fortheelectrostaticandvan derWaalsterms. Thetemperaturecouplingsectionisveryimportantandmustbefilledoutcorrectly. Tcoupl =berendsen [8](typeoftemperaturecouplinganotheroptionisnosehoover) tau_t=Timeconstantfortemperaturecoupling(units=ps).Youmustlistoneper tc_grpinthe orderinwhichtc_grpsappear. tc_grps=groupstocoupletothethermostat(everyatomorresidueinyourmodelmustbe representedherebyappropriateindexgroups). ref_t=referencetemperatureforcoupling(i.e.thetemperatureofyourMDsimulationindegrees K).Youmustlistonepertc_grpintheorderinwhichtc_grpsappear. Whenyoualterthetemperature,dontforgettochangethegen_tempvariableforvelocity generation. pcoupl istheParrinelloRahmanbarostat. pcoupltypeisotropicmeansthattheboxwillexpandorcontractevenlyinalldirections(x,y, andz)inordertomaintaintheproperpressure. Note:Usesemiisotropicformembranesimulations. tau_p timeconstantforcoupling(inps). 1 compressibilitythisisthecompressibilityofthesolventusedinthesimulationinbar (the settingaboveisforwaterat300Kand1atm). ref_pthereferencepressureforthecoupling(inbar)(1atm~0.983bar). 8

9. GROMACSTutorial Preprocessthepr.mdpfile. gromppf pr.mdpcem.grop fws.top opr.tpr nohupmdrundeffnm pr& Usethetail commandtocheckthepr.logfile. Themd.mdpparameterfilelooksvery similartothepr.mdpfile.Thereareseveraldifferences. Thedefinestatementisnotnecessaryaswearenotrunningaspositionrestraineddynamics. Contentofmd.mdpforexplicitsolvation (SpecialNote:Forinvacuo,removetheentriesforsol inthetemperaturecouplingsection.Forthesimulationthatincludesthecounterions,addanentry fortheionsinthetemperaturecouplingsection.) title=FWS cpp=/usr/bin/cpp constraints=allbonds integrator=md dt=0.002 ps! nsteps=25000 total50ps. nstcomm= 1 nstxout=500 collectdataevery1ps nstvout=0 nstfout=0 nstlist=10 ns_type=grid rlist=1.0 coulombtype =PME rcoulomb=1.0 vdwtype =cutoff rvdw=1.4 fourierspacing =0.12 fourier_nx =0 fourier_ny =0 fourier_nz =0 pme_order =4 ewald_rtol =1e5 optimize_fft =yes Berendsentemperaturecouplingison Tcoupl=berendsen tau_t =0.1 0.1 tcgrps =protein nonprotein ref_t =300 300 Pressurecouplingison Pcoupl=parrinellorahman tau_p=0.5 compressibility=4.5e5 ref_p=1.0 Generatevelocitesisonat300K. gen_vel=yes 9

10. GROMACSTutorial gen_temp=300.0 gen_seed=173529 gromppfmd.mdpcpr.grop fws.top omd.tpr nohupmdrundeffnmmd& Usethetail commandtocheckthemd.logfile. Youmaycompressthetrajectoryusingtrjconvtosaveondiskspace. trjconvfmd.trr omd.xtc Onceyouhavemadethe*.xtcfile,youmaydeletethe*.trrfile. Usengmx toviewthetrajectory(youmayalsodownloadtoyourPCanduseVMDtoviewthe trajectory). ngmx fmd.trr(ormd.xtc)smd.tpr Whentheviewercomesup,youwillseeadialogboxwithanumberofselections.Checkthebox labeledProtein,thenclickOk. ngmxsinitialstartupdialog. 10

11. GROMACSTutorial SelectingProteinpermitsustoviewtheproteinonlywithoutinterferencefromthe~3,000water moleculesthatoccupytherestofthebox. UseXRotate torotatetheboxupanddown(leftmouseclickgoesuprightmouseclickgoes down).UseYRotate torotatetheboxfromlefttorightorviceversa(leftmouseclickrotatesto theleftandrightmouseclickrotatestotheright). Scaleatthebottompermitsyoutozoominand zoomout(useleftmousebuttontozoominandrightmousebuttontozoomout). ToviewanothergroupinthemodelgotoDisplay>Filterandtheinitialdialogboxwillcome upagainandyoumayopttoviewsomeotherindexgroup(e.g.thebackbone). ToviewtheanimationoftheMDtrajectorygoto Display>Animate.Controlsusedforthe playbackofthetrajectoryappearatthebottomofthewindow.Usethecenterarrowbuttontoview onetimestepattime.Usetheforwarddoublearrowtoplaythewholetrajectory,andusethe pausebuttontotherighttostopthetrajectoryplayback.Usethedoublearrowtothelefttoreset theanimation. Unfortunately,thesaveaspdboptionundertheFilemenuinngmxdoesnotwork.Therefore Thebestwaytoviewthetrajectoryandselectsnapshotstosaveas*.PDBfilesistousevisual moleculardynamics(VMD). (Downloadfrom:http://www.ks.uiuc.edu/Research/vmd/ Itsfreeto academicsanditrunsonunixandwindows!). Analysis OneofthemajoradvantagesofGromacs(otherthanthefactthatitisGNUpublicdomainand free!)istherobustsetofprogramsavailableforanalyzingthetrajectories.Wewilldiscussafewof themostimportantanalytictoolsintheGromacsarsenalhere. 11

12. GROMACSTutorial Groups make_ndx Themake_ndxprogramisusefulforgeneratinggroups(IDtagsforspecificatomsorresiduesthat youmaywanttoanalyze).Gromacsgeneratesdefaultgroupswhichmaybeadequateforyourwork asis.However,ifyouneedtodomoreindepthanalysis,usemake_ndxtotagspecificitemsin yourmodel.Seethemanualformoreinformation. Howtousemake_ndx tosetupindex(ndx)files.Usemake_ndxtoidentifyparticulargroupsthat youmightwanttofreezeduringasimulationorgatherspecialenergyinformation.Letslookatan examplewherewewanttofreezetheNandCterminalaminoacidsofaprotein.Alwaysuse make_ndxtocreateanindexgroupforusewiththegromppprogram. Inthissamplecase,wehaveacoordinatefileofacollagentriplehelix,postpositionrestrained dynamics,wherewewanttofreezetheN&Cterminalforproductionrun.Wemustfirstidentify theresidue#sinthecoordinatefile(Inourcaseclg_b4md.pdb)thatidentifytheN&Ctermini. Thecommandlineissimpleenough make_ndx f clg_b4md.pdb oclg_ter.ndx Youwillseethefollowingoutput(weleftoutthedescriptiveinfoatthebeginning)followedbya commandprompt(>). Readingstructurefile Goingtoread0oldindexfile(s) Analysingresiduenames: Openinglibraryfile/usr/share/gromacs/top/aminoacids.dat Thereare: 2194OTHERresidues Thereare:108PROTEINresidues Thereare:0DNAresidues AnalysingProtein... AnalysingOther... 0System:7365atoms 1Protein:765atoms 2ProteinH:687atoms 3Calpha:108atoms 4Backbone:324atoms 5MainChain:435atoms 6MainChain+Cb:507atoms 7MainChain+H:477atoms 8SideChain:288atoms 9SideChainH: 252atoms 10ProtMasses:765atoms 11NonProtein:6600atoms 12DRG:21atoms 13SOL:6579atoms 14Other:6600atoms 12

13. GROMACSTutorial nr:group!'name'nrname'splitch'nr Enter:listgroups 'a':atom&'del'nr'splitres'nr'l':listresidues 't':atomtype|'keep'nr'splitat'nr'h':help 'r':residue'res'nr'chain'char "name":group'case':casesensitive 'q':saveandquit > UsethercommandtoenterthelistofresiduenumbersthatrepresenttheN&Cterminiofthe triplehelix. >r136377273108 15r_1_36_37_72_73_108:51atoms Note:Youmayalsouseadashtospecifyaresiduenumberrange(e.g.tospecifyresidues1to36 use>r136) Thedefaultname(r_1_36_37_72_73_108 ) givingtothenewindexgroupthatyouhavejust creatediscumbersome.Letsrenameitusingthenamecommand.Wewillusetheindexgroup# (15)inthecommand. >name15Terminal >v Turnedverboseon 0System:7365atoms 1Protein:765atoms 2ProteinH:687atoms 3Calpha:108atoms 4Backbone:324atoms 5MainChain:435atoms 6MainChain+Cb:507atoms 7MainChain+H:477atoms 8SideChain:288atoms 9SideChainH:252atoms 10ProtMasses:765atoms 11NonProtein:6600atoms 12DRG:21atoms 13SOL:6579atoms 14Other:6600atoms 15Terminal:51atoms nr:group!'name'nrname'splitch'nrEnter:listgroups 'a':atom&'del'nr'splitres'nr'l':listresidues 't':atomtype|'keep'nr'splitat'nr'h':help 'r':residue'res'nr'chain'char "name":group'case':casesensitive'q':saveandquit > 13

14. GROMACSTutorial Weusedthevcommandtoverifythatournamechangewassuccessful.Tofinishup,usetheq commandtosaveandquitandyouredone. Nowhowdowefreezethegroups?Easy,addthefollowinglinestoyourmd.mdpfile energygrps_excl=TerminalTerminalTerminalSOL!Toremove computationofnonbondinginteractionsbetweenthefrozengroupswitheachother andsurroundings(i.e.thesolvent,SOL) freezegrps =Terminal!Indexgrouptofreeze freezedim =YYY !Freezethisgroupinalldirections,x, y,andz Remember,youmustincludethenewindexfilewhenyouusethismd.mdpfiletocreateyourrun inputfile(tpr)usinggrompp.Usethenflagtogrompp.Forexample gromppfmd.mdpcpr.grop clg.top n clg_ter.ndx omd.tpr Properties g_confrms TocomparethefinalstructuretotheoriginalPDBfileobtainedfromthePDBuseg_confrms(fora descriptionuseg_confrmsh).Thisprogramtakestwostructuresandperformsaleastsquaresfit. g_confrmsf11OMB.pdb f2md.groofit.pdb Youwillbepromptedtoselectagroup(selecttheBackbone(group4)bothtimes).Theprogram reportsanRMSdeviationandproducesanoutputfile(fit.pdb).Theoutputfilewillshowthetwo structuressuperimposedon eachother. g_covar Usetocomputethecovariancematrix(seemanualformoreinfo).Mayalsobeusedtocomputean averagestructurefromadynamicstrajectory.Forexampletocomputetheaverageofthelast200 psofa1nsdynamicsrunuse g_covar f traj.xtcstopol.tprb 801e 1000av traj_avg.pdb WarningAveragestructurestendtobecrude.Minimizationisrecommended. g_energy Usethisprogramtoplotenergydata,pressure,volume,density,etc. g_energyfmd.edrofws_pe.xvg 14

15. GROMACSTutorial Youwillbepromptedforthespecificdatathatyouwanttoincludeintheoutputfile(*.xvg).The outputfileisatypeofspreadsheetfilethatcanbereadusingXmgrorGrace.Thisfileisatextfile andcanbereadintoMicrosoftExcelhowever,youwillneedtodosomelighteditingofthefile. Forexampleinourcaseabove(yourresultsmaybedifferent),weseethefollowing Selectthetermsyouwantfromthefollowinglist G96AngleProperDih.ImproperDih.LJ14Coulomb14 LJ(SR)LJ(LR)Coulomb(SR)Coul.recip.Potential KineticEn.TotalEnergyTemperaturePressure(bar)BoxX BoxYBoxZVolume Density(SI)pV VirXXVirXYVirXZVirYXVirYY VirYZVirZXVirZYVirZZPresXX(bar) PresXY(bar)PresXZ(bar)PresYX(bar)PresYY(bar)PresYZ(bar) PresZX(bar)PresZY(bar)PresZZ(bar)#Surf*SurfTenPcouplMuXX PcouplMuYYPcouplMuZZMuXMuYMuZ TProteinTSOLTCL LambProteinLambSOL LambCL Forpotential energy,typePotential Hitthekeyagain WegetasummarywithaveragePEandRMSDinkJ/mol Lastframeread500time100.000 Statisticsover50001steps[0.0000thru100.0000ps],1datasets Energy AverageRMSDFluct.DriftTotDrift Potential 192661311.547311.5010.1846818.4684 Toread*.xvgfilesintoGraceusethefollowingcommand: xmgracenxyfws_pe.xvg YoumayobtainGracefrom http://plasmagate.weizmann.ac.il/Grace/.GracerunsonLinuxandin Unixonly. IfyoudonothaveGraceorXmgr,justimportorreadthe*.xvgfileintoMSExcelasa spacedelimitedfile. g_gyrate Useg_gyratetomeasuretheradiusofgyration.Thisquantitygivesameasureofthe compactnessofthestructure.Thisgivesameasureofthemassoftheatom(s)relativethecenter ofmassofthemolecule. g_gyratefmd.trrsmd.tpr ofws_gyrate.xvg g_rmsandg_rmsdist 15

16. GROMACSTutorial Theseprogramsareusefulformeasuringrootmeansquaredeviationsinthestructure.Useg_rms toevaluatethedeviationofthestructurefromtheoriginalstartingstructureoverthecourseofthe simulation.(Thedt10optiontellstheprogramtowriteeverytenthframe) g_rmss*.tpr f *.xtc dt 10 TocomparermsdtotheNMRstructureusethefollowingcommand g_rmssem.tprf md.trro fws_rmsd.xvg Selectgroup4(Backbone)fortheleastsquaresfit.Theprogramwillgenerateaplotofrmsdover time(rmsd.xvg)whichyoumayimportintoExcelasaspacedelimitedfile. g_rmsf Computesrootmeansquarefluctuationofatompositions.Likeg_covar thiscommandmaybe usedtocomputeaveragestructures.Forexample,tocalculatetheaverageofthelast500psofa2 ns(2000ps)dynamicssimulation,usethefollowing g_rmsf f traj.xtcs topol.tpr b1501 e2000 o traj_rmsf.xvgox traj_avg.pdb SelectarangewhereyouobservesomeequilibriumfromyourRMSDplot(computedusing g_rms).Forexample Theexampleaboveistheresultofa1nssimulation (yourresultsmightbedifferent).This simulationstillneedstoberunevenlongerasithasnotcompletelyequilibrated. Intheexample above,wemightuse the200to500 psrangeforcomputinganaveragestructureasweobservea levelingoffindeviationfromthereference(initialstructure). Thecommandwouldbe g_rmsf smd.tprf md.trrb 200e 500ox fws_avg.pdb 16

17. GROMACSTutorial Selectgroup1Proteinwhenprompted. WarningAveragestructurestendtobecrude.Minimizationisrecommended. Suggestedem.mdpfilessetupforan invacuominimization.Performsteepestdescentsfirst followedbyconjugategradient. CAUTION!Youmayneedtorun pdb2gmx tosetupanewsetof topologyfilesforyourminimizationespeciallyifyouhaveisolatedaspecificgroupincomputing youraveragestructure(asopposedtotheentiresystem). SteepestDescentsinvacuo Userkerrigan(236) Inputfile cpp=/usr/bin/cpp constraints=none integrator=steep nsteps=400 Energyminimizingstuff emtol=1000 emstep =0.01 nstcomm=1 ns_type=grid morse=no coulombtype=Shift vdw_type=Shift rlist=1.4 rcoulomb=1.2 rvdw=1.2 rcoulomb_switch= 1.0 rvdw_switch=1.0 epsilon_r=6.0 Tcoupl=no Pcoupl=no gen_vel=no Conjugategradientwithmorsepotential invacuo Userkerrigan(236) Inputfile cpp= /usr/bin/cpp constraints=none integrator=cg nsteps=3000 Energyminimizingstuff emtol=100 emstep=0.01 17

18. GROMACSTutorial nstcgsteep=1000 nstcomm=1 morse= yes coulombtype=Shift vdw_type=Shift ns_type=grid rlist=1.4 rcoulomb=1.2 rvdw=1.2 rcoulomb_switch=1.0 rvdw_switch=1.0 epsilon_r=6.0 Tcoupl=no Pcoupl=no gen_vel=no Theg_rmsf commandmayalsobeusedtocomputetemperaturefactors.Thecomputed temperaturefactorscanbecomparedtoxraycrystal structure temperaturefactors. g_rmsf smd.tprf md.xtcormsf.xvgoqfws_bfac.pdb UsetheBackbonegrouponceagain. 100 90 80 TemperatureFactor 70 60 50 40 30 20 10 0 0 50 100 150 200 250 300 Residue# do_dssp Usethedo_dsspcommandtoanalyzethesecondarystructureofyourmodel.Youmusthavethe dsspprogram (http://swift.cmbi.ru.nl/gv/dssp/)installed(in/usr/local/bin).[9] do_dsspsmd.tpr f md.xtcofws_ss.xpm SelectGroup1(Protein)forthecalculation.Usethexpm2psprogram toconvertthexpmfiletoeps format.ThenusetheImageMagickconvertprogramtoconverttheepsfiletopngorwhatever desiredimageformat. 18

19. GROMACSTutorial xpm2psffws_ss.xpm ofws_ss.eps convertfws_ss.epsfws_ss.png Whereresidue#isontheyaxisandtime(ps)isonthexaxis.Asseenfromtheoriginal NMR structurebelow Fromthedsspplotabove,wesee3regionsof redforthe3sheets(displayedinyellowinthe structureimagetotheleft).Theoneshort regioninthemiddleistheleaststable.The imagetotheleftwaspreparedusingthePymol program.(http://pymol.sourceforge.net/ ) g_hbond Useg_hbond tomeasurethenumberofhydrogenbonds,hbonddistances&anglesbetween moleculesorgroupsthroughthecourseofthesimulation. g_hbondf md.trrsmd.tpr num fws_hnum.xvg H a r D A Geometricalrelationsusedbyg_hbond. ThedefaultsinGromacs3.3.1are: r 0.35 nm a 30 19

20. GROMACSTutorial Usether andaflagstosetotherlimits.Bydefault,g_hbondanalyzesthedonoracceptor(rDA) distance). Youmaychangethisbehaviourbysettingthedaflagtono.Settingdatonoinstructs g_hbondtoanalyzetherHA distance. SeetheGromacsmanualformoreoptions. g_saltbr Useg_saltbr toanalyzesaltbridgesbetweenresiduesinyoursimulation. Theprogramoutputsa setof*.xvgfiles,whichgivedistancesbetweenminus/minus,chargedresiduesplus/minus(the mostinteresting)andplus/pluschargedresidues. g_saltbr fmd.trrsmd.tpr HowToSavespecifictimepointsfromatrajectoryas*.PDBfiles: Togetaspecificframe(3000psinthisexample)insteadofthewholetrajectoryasapdbfileuse additionallythedumpoption,e.g. trjconvf traj.xtcsfile.tpro time_3000ps.pdb dump3000 20

21. GROMACSTutorial Performotheranalysistasksasrecommendedbyyourinstructor. Appendix. Howtorestartacrashedrun. tpbconv sprev.tpr f prev.trr eprev.edro restart.tpr mdrunsrestart.tpr deffnmmyrestart (thedeffnm optionsetsthedefaultfilenameforallfile optionstomdrun) Howtoextendarun. tpbconv f traj.trr s topol.tpr eener.edr o tpxout.tpr time$VALUEuntil $VALUE Where$VALUE=ps(e.g.ifyouwantedtoextenda2nsrunto5ns,then$VALUE=5000) Howtosetupaparallelrun gromppnp#f md.mdpcpr.gropfws.topo md.tpr Wherethenpflagisusedtodesignatethenumberofnodesforparallelexecution. Thenusemdrun_mpitoruntheparalleljob.ForexampleontheUMDNJSunFireuse mprunnp#/products/gromacs/sparcsunsolaris2.8/bin/mdrun_mpideffnmmd UsetheabovecommandinyourSunGridEnginebatchsubmissionscript! Bibliography 1. Lindahl,E.,B.Hess,andD.vanderSpoel,GROMACS3.0:apackageformolecular simulationandtrajectoryanalysis.J.Mol.Model,2001.7:p. 306317. 2. Berendsen,H.J.,J.Grigera,andT.Straatsma,Themissingtermineffectivepairpotentials. J.Phys.Chem.,1987.91:p.62696271. 3. Hess,B.andN.vanderVegt, Hydrationthermodynamicpropertiesofaminoacid analogues:asystematiccomparisonofbiomolecularforcefieldsandwatermodels.J.Phys. Chem.B,2006.110(35):p.1761626. 4. Weber,W.,P.H.Hnenberger,andJ.A.McCammon,MolecularDynamicsSimulationsofa PolyalanineOctapeptideunderEwaldBoundaryConditions:InfluenceofArtificial PeriodicityonPeptideConformation.J.Phys.Chem.B,2000.104(15):p.36683575. 5. Darden,T.,D.York,andL.Pedersen,ParticleMeshEwald:AnNlog(N)methodforEwald sumsinlargesystems.J.Chem.Phys.,1993.98:p.1008910092. 6. Essmann,U.,L.Perera,M.L.Berkowitz,T.Darden,H.Lee,andL.Pedersen,Asmooth particlemeshewaldpotential.J.Chem.Phys.,1995.103:p.85778592. 21

22. GROMACSTutorial 7. Hess,B.,H.Bekker,H.Berendsen,andJ.Fraaije,LINCS:ALinearConstraintSolverfor molecularsimulations.J.Comp.Chem.,1997.18:p.14631472. 8. Berendsen,H.J.C.,J.P.M.Postma,W.F.vanGunsteren,A.DiNola,andJ.R.Haak, Moleculardynamicswithcouplingtoanexternalbath.J.Chem.Phys.,1984. 81(8):p. 35843590. 9. Kabsch,W.andC.Sander,Adictionaryofproteinsecondarystructure.Biopolymers,1983. 22:p.25772637. 22