AMBER9.

0Drug/DNAComplexTutorial

JohnE.Kerrigan,Ph.D. RobertWoodJohnsonMedicalSchool TheUniversityofMedicineandDentistryofNewJersey 675HoesLane Piscataway,NJ08854 USA (732)2354473phone (732)2355252fax kerrigje@umdnj.edu

Synopsis.Todayyouwilllearnhowtouseoneofthemostversatilemoleculardynamics modelingpackages,AMBER9.[1] TheAMBERsuiteofprogramswasdevelopedbythelate PeterAKollmanandcolleaguesattheUniversityofCaliforniaSanFrancisco(UCSF)andis nowmaintainedatScrippsInstitute.SeetheAmberwebpageathttp://amber.scripps.edufor moreinformation.TheAmberpackagewasdesignedwiththeabilitytoaddressawidevariety ofbiomoleculesincludingproteinsandnucleicacidsaswellassmallmoleculedrugs. ResearchProblem.WewillworkonthecomplexofaDNAminorgroovebindingdrug(goxa pentamidine).Wewilltakethexraycrystalstructure(166D.PDB)[2]ofthecomplex,andstudy itsbehaviorinwaterusingmoleculardynamics.Makeanewprojectdirectoryusingthemkdir command.Copy166D.pdbandpet.pdbtothisdirectory. Preparationofthedrug.Openyourpet.pdbfileinMOE. OpenMOE.GotoFile>Open>pet.pdb(ClickonAutoConnectandCenterinthedialog).
H N H N H O O O H N H N H

OpentheBuilderbyClickingontheBuilderbuttonontherighthandsideoftheMOE window.Changetheformalchargeofoneofthenitrogensofeachaminidiniumgroupto+1(i.e. onenitrogenoneachsideofthemolecule).UsetheElement:toolsin MoleculeBuilder. Edit>AddHydrogens IMPORTANT:Eachatomofthedrugmoleculemusthaveauniquename!MOEdoesnotdo thisautomatically!ClickonLabels>Nametodisplaytheatomnames.SelecteachHydrogen atomonebyoneandgiveitauniquenameusingtheatomsdialog.Selecttheatomwithyour mouse(turnspink)then ClickonAtoms(righthandsidemenu)>AtomManagerClickon Selectiononly.HighlightthehydrogenatomintheAtomManagerandchangethenamefromH toH#(where#isthenextnumberinthesequenceexaminetoseeiftheotherhydrogenatoms arenumberedotherwisestartnumberingfrom1.).Whenyouarefinished,replacetheexisting filewiththenewfile. File>SaveAs>FileFormat:PDB:pet.pdb ThetablebelowshowstheAMBERnamesfornucleicacidresidues.ThosenameswithaDare forDNA residuesandthosewithanRareforRNAresidues.Aterminal5 residuewouldbe indicatedbye.g.DA5anda3 residuewouldbee.g.DT3.However,Leapwillrecognizethe normalnucleicacidnamesgivenandassumethemtobeDNA.Leapisabletodifferentiate3 from5 residues.

GrouporResidue Adenine Thymine Uracil Cytosine Guanine

ResidueName,Alias DA,RA DT RU DC,RC DG,RG

Open166D.pdbinneditandopenpet.pdbinnedit.ReplacetheoldPETresiduecoordinate recordswiththenewPETcoordinates.Savethenewfileasdna_pet.pdb. Makingaspecialtopologyprepinputfiletodescribethedrug.AMBERhasaresidue topologydatabasetodescribeallofyournucleicacidresiduesaswellasaminoacidresiduesif youhappentobeworkingwithaprotein.However,AMBERdoesnothaveaspecial topology databasetodescribeyourdrug.Fortunately,AMBERhasaspecialprogramcalled antechamber,whichisdesignedtobuildatopologyprepfileforyourdrug. [3] In combinationwith DivCon (asemiempiricalQMprogram),antechamberwillcomputepartial atomicchargesforyourdrug.WewilluseAM1BCCcharges[4],asthesechargesscalebest withthechargesetwewillbeusingonourDNA.Bydefault,antechamberwillwriteaprepfile withthegeneralamberforcefield(gaff)atomtypes.TowriteaprepfilewithregularAmber atomtypes,usetheatamberflag.However,alwaysusethegaffforcefieldforyoursmall molecules.Issuethefollowingcommandfromwithinyourworkingdirectory.
antechambernc2rnPETipet.pdbfipdbopet_bcc.prepfoprepicbcc

Whatthecommandlineflagsmean: nc rn i fi o fo c isthenetmolecularcharge(veryimportant!) istheresiduename(defaultisMOL)Useanameherethatyouknowisusedinyourpdb file! istheinputfile istheinputfiletype(e.g.pdb,mol2seeAMBERmanualformoreinfo) istheoutputfilename istheoutputfiletype(e.g.prepiforAMBERprepfile) isthechargetypeused(bccwillcomputeAM1BCCcharges rcwillreadincharges fromtheinputfileseeAMBERmanualformoreinfo)

Contentsoftheprepfile
002 Thisisaremarkline molecule.res PETXYZ0 CORRECTOMITDUBEG 0.0000 1DUMMDUM0 1 20.000.0.0.00000 2DUMMDU M10 11.449.0.0.00000 3DUMMDUM2101.522111.1.0.00000 4N1nh M3211.540111.208180.000 0.456 5H4hnE4321.01059.922 148.3840.334

etc
# nametype ts connectivity bndlen angle dihedral charge LOOP (Describering(i.e.cyclic)structures) C1C6 C13C8 IMPROPER (Improperdihedrals) C4N1C7N2 C7C5C4C3 C4C6C5H5 C5C1C6H6 C4C2C3H7 C3C1C2H8 C6C2C1O1 C9C13C8O2 C8C10C9H17 C9C11C10H18 C14C10C11C12 C11N3C14N4 C11C13C12H20 C8C12C13H19 DONE STOP

#atomnumberforatomI(canbeanyatom) nameatomnameforatomI typeatomtypeforatomI tsTreeStructure Describesthegeometryofthestructureandhowitlinkstotherestofthe largerstructureifthisprepfiledescribesaresidue.Thereare5topologicaltypes:Main,Side, Branch,(3,4,5,&6),andEndor M, S, B,3etc., E. Mainatomsdescribethepaththroughtheresidueconnectingittothenextresidue. EisfortheEndatomofachain.TheEatomscanonlyhaveoneconnectiontoanotherheavy atom. S (side)musthaveconnectionstotwootheratoms. B(branch)musthaveconnectionsto threeotherheavyatoms.A3atomhasatotaloffourconnectionstoheavyatoms(aquaternary atom).The4,5,and6areusedtodescribehigherorderbonding(e.g.metalcomplexes). LOOPclosingsectiondescribescyclicsystems.Loopconnectionsarenotcountedwhen assigningM,S,B,etc.

DUMMYATOMSThreearerequired.Theseareusedtodefinethespaceaxesfortheresidue. TheymustbegiventhetopologicaldescriptionofM,andmustbeoftheDummyatomtype, DU. connectivity Hereiswhatthe3numbersmeaninreferencetoatomI: NA(I) NB(I) NC(I)

st 1 numberTheatom#towhichatomIisconnected. nd 2 numberTheatom#towhichatomImakesananglealongwithNA(I) rd 3 numberTheatom#towhichatomImakesadihedralwithNA(I)&NB(I).

bndlen EquilibriumbondlengthbetweenatomIandNA(I). angleThebondanglebetweenNB(I),NA(I)andI. dihedral ThedihedralanglebetweenNC(I),NB(I),NA(I),andI chargeThepartialatomicchargeonI. LOOPDescribesaloopclosingbondforeachcyclicringinthemolecule. IMPROPERTellsamberwhichimpropertorsionanglesaretobeusedforthecalculation.In unitedatommodels,impropertorsionsareusedtokeepasymmetriccentersfromracemizing.In additionimpropertorsionsareusedtoenforceplanarityincyclicringsystemsinbothunitedand allatommodels.Theimpropertorsionsaretobedefinedinsuchawaythatthepropertorsions arenotduplicated.

Useparmchk tochecktheparameters.
parmchkipet_bcc.prepfprepiopam.frcmod

Checkthepam.frcmodfilecontents(usemorepam.frcmod).Youwillnoticethatthereareafew parametersthatneededestimatesbasedonexistingparameters.Theoutputhasnowarnings aboutunknownatomsorparameters.Therefore,wemayproceed. Contentsofthepam.frcmodfile:

remarkgoeshere MASS BOND ANGLE nhcenh72.900117.500sameasnhc2nh nhceca66.206117.715Calculatedwithempiricalapproach DIHE nhcenhhn10.675180.0002.000sameasXc2nhX nhcecaca12.550180.0002.000sameasXc2caX hnnhceca10.675 180.0002.000sameasXc2nhX

IMPROPER canhcenh1.1180.02.0Usingdefaultvalue cacacace1.1180.02.0Usingdefaultvalue cacacaha1.1 180.02.0Generalimpropertorsional angle(2generalatomtypes) cacacaos1.1180.02.0Usingdefaultvalue NONBON

xLeapandtLeap Theseprogramsperformthesamefunctionwiththedifferencebeingthat xLeapopensinanxwindowinterfaceandtLeapoperatesfromaterminalprompt(noGUI). TheprincipalfunctionoftheseprogramsistopreparetheAMBERcoordinate(inpcrd)and topology(prmtop)files.

xleapsfleaprc.ff99 AdjustforuseoftheSPCE/Ewatermodel. HOH=SPC WAT=SPC loadamberparamsfrcmod.spce

Theleaprc.ff99fileloadstheparametersfortheAMBER99forcefield.[5,6]Theleaprc.gaff fileloadstheparametersetforthegeneralamberforcefield, whichisusedtodescribeyour smallmolecule. Intheleapwindoworprompttypethefollowingcommandsoneatatime:

sourceleaprc.gaff loadAmberParamspam.frcmod loadAmberPreppet_bcc.prep nap=loadPdbdna_pam.pdb checknap

Thecheckrevealsawarningforthechargeof theunitasbeingnonzero.Wewilladd counterionslatertoneutralize.

editnap

Thefollowingmousebuttonsperformasindicated: LEFT MIDDLE RIGHT MIDDLE+RIGHT Selection Rotate Translate ZoomInandOut

Thestructureshouldbefineatthispoint.YoushouldnoticethatLeaphasaddedhydrogen atomstotheDNAstructure.ToexittheuniteditoruseUnit>closefromthemenu. SolvatethestructureinSPC/Ewater [7]usingacubicperiodicbox.Usethefollowing command

solvateBoxnapSPCBOX9.0

YouhavetoldLeaptosolvatetheunitinacubicboxusingaspacingdistanceof9.0angstroms aroundthemolecule.Ideally,youshouldsetthespacingatnolessthan8.5(~3waterlayers) toavoidperiodicityartifacts.[8] Forparticlemeshewaldelectrostatics,[9,10]ourboxside lengthmustbe>2XNBcutoff.Wewillusea9.0 cutofftherefore,ourboxsidemustbe> 18.Ourboxsidelengthwillbe(2X8)+DNAdimension,whichshouldbegreaterthan18. Now,letsaddcounterionstoneutralizethecharge.Wehadatotalchargeof 20.000therefore, weneed20sodiumionstoneutralize.
addIonsnapNa+0

Thecommandwejustissued(addIons)addedNa+ionsuntilthetotalnetformalcharge=0. Thisshouldadd20Na+ions.Nowwecansavethetopologyandcoordinatefiles!
saveAmberParmnappet.toppet.crd

Asanadditionalstep,youmaywanttosaveaPDBfileofthemodelyoujustcreated.Todo this,justusethesavePdbcommand.
savePdbnappet_na.pdb

Youredonewithfilepreparation(hooray!).TypeQuittoexitxLeap! UseRasmol orPymol toviewyourmodel.Inthewinterm,typerasmol pet_na.pdb. Our modelbeforedynamics

MolecularDynamics
Welldothesecomputationsin4steps.Thefirstthreestepsarepreparatoryandthelaststepis whatisknownastheproductionrun. Step1. EnergyMinimizationoftheSystem.Weperformasteepestdescentsenergy minimizationtorelievebadstericinteractionsthatwouldotherwisecauseproblemswithor dynamicsruns. UsingtheSanderprogram.TheSANDERprogramisthenumbercrunchingjuggernautofthe AMBERsoftwarepackage.SANDERwillperformenergyminimization,dynamicsandNMR refinementcalculations.YoumustspecifyaninputfiletotellSANDERwhatcomputationsyou wanttoperformandhowyouwouldliketoperformthosecomputations.Studytheinputfilefor energyminimizationbelow. Thecontentsofthemin1.infile.
InitialrestrainedminimizationonDNA&DRG,10cut &cntrl imin=1, maxcyc=1000, ncyc=250, ntb=1, ntr=1, cut=10 / HoldtheDNA&DRGfixed 500.0 RES125 END END

Whatitallmeansinanutshell. FormoreindepthinfopleaseconsulttheAMBERUsers Manual.Thesettingsimportantforminimizationarehighlightedandexplainedbelow. &cntrl and/Mostifnotallofyourinstructionsmustappearinthecontrolblock(hence &cntrl). cut=nonbondedcutoffinangstroms. ntr =Flagusedtoperformpositionrestraints(1=on,0=off) imin=Flagtorunenergyminimization(if=1thenperformminimizationif=0thenperform moleculardynamics). macyc=maximum#ofcycles ncyc=Afterncyccyclestheminimizationmethodwillswitchfromsteepestdescentsto conjugategradient. ntmin=Flagforminimizationmethod.(if=0thenperformfullconjugategradientminwiththe first10cyclesbeingsteepestdescentandeverynonbondedpairlistupdateif=1forncyccycles

steepestdescentisusedthenconjugategradientisswitchedon[default]if=2thenonlyuse steepestdescent) dx0=Theinitialsteplength dxm =Themaximumstepallowed drms=gradientconvergencecriterion1.0E4kcal/molisthedefault HoldtheDNAandtheDRGfixed 500.0 (Thisistheforceinkcal/molusedtorestraintheatompositions.) RES125 (TellsAMBERtoapplythisforcetoresidue#s1to25). Commandlineingeneral(Exampleonly):
sanderOimin.inomin.outpprmtopcinpcrdrrestrt[refrefcx mdcrdvmdvelemdeninfmdinfo]

Issuethefollowingcommandtorunyourenergyminimization.
nohupsanderOimin1.inomin1.outppet.topcpet.crdrpet_min1.rst refpet.crd&

Monitortheprogressusingtail 20min1.outcommand. Step2.Minimizationofwholesystem,norestraints. Contentsofmin2.infile

SystemminimizationonDNA&DRG,10cut &cntrl imin=1, maxcyc=1500, ncyc=500, ntb=1, ntr=0, cut=10 / END nohupsanderOimin2.inomin2.outppet.topcpet_min1.rstr pet_min2.rst&

Step3. PositionRestrainedDynamics.Thisinitialdynamicsrunisperformedtorelaxthe positionsofthesolventmolecules.Inthisdynamicsrun,wewillkeepthemacromoleculeatom positionsrestrained(notfixed,however).Inapositionrestrainedrun,weapplyaforcetothe specifiedatomstominimizetheirmovementduringthedynamics.Thesolventweareusingin oursystem,water,hasarelaxationtimeof10psthereforeweneedtoperformatleast>10psof positionrestraineddynamicstorelaxthewaterinourperiodicbox. Contentsofmd1.in

positionrestraineddynamics,model1,10.0cut &cntrl imin=0,

irest=0, ntx=1, ntb=1, cut=10, ntr=1, ntc=2, ntf=2, tempi=0.0, temp0=300.0, ntt=3, gamma_ln=1.0, nstlim=12500,dt=0.002, ntpr=100,ntwx=500,ntwr=1000 / HoldthedrugandDNAfixed 10.0 RES125 END END

ntb=1 Constantvolumedynamics. imin=0Switchtoindicatethatwearerunningadynamics. nstlim =#ofstepslimit. dt=0.002timestepinps(2fs) temp0=300referencetemp(indegreesK)atwhichsystemistobekept. tempi=0initialtemperature(indegreesK) ig=###seedforrandomnumbergenerator heat=0multiplierforvelocities(default=0.0). ntt=3 temperaturescalingswitch(1=LangevinDynamics) 1 gamma_ln=1.0collisionfrequencyinps whenntt=3(seeAmber8manual). tautp =1.0Timeconstantfortheheatbath(default=1.0)smallerconstantgivestighter coupling. vlimit=20.0usedtoavoidoccasionalinstabilityindynamicsruns(velocitylimit20.0isthe default).Ifanyvelocitycomponentis>vlimit,thenthecomponentwillbereducedtovlimit. comp=44.6unitofcompressibilityforthesolvent(H2O) ntc=2FlagfortheShakealgorithm(1NoShakeisperformed2bondstohydrogenare constrained3allbondsareconstrained). tol =#.#####relativegeometrictoleranceforcoordinateresettinginshake. Theadditionalblocksthatfollowarethenmrrestraintinstructions.Wewillgraduallywarmthe systemfrom100Kto300Kinthefirstpicosecondfollowedbymaintainingthetemperatureat 300K. Youwillnotethatweusedasmallerrestraintforce(10.0kcal/mol).Fordynamics,oneonly needstouse5to10kcal/molrestraintforcewhenntr=1(usesaharmonicpotentialtorestrain coordinatestoareferenceframehence,theneedtoincludereferencecoordinateswiththeref flag.).Largerrestraintforcesleadtoinstabilityintheshakealgorithmwitha2fstimestep. Largerrestraintforceconstantsleadtohigherfrequencyvibrations,whichinturnleadtothe instability.Excessmotionawayfromthereferencecoordinatesisnotpossibleduetothe steepnessoftheharmonicpotential.Therefore,largerestraintforceconstantsarenotnecessary.

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nohupsanderOimd1.inomd1.outppet.topcpet_min2.rstr pet_md1.rstxpet_md1.mdcrdrefpet_min2.rstinfmd1.info&

Monitortheprogressusingtail 40md1.out.Ourcomputationtookabout1.8 hoursto completeona2.7GHzlinuxworkstation. Step4. TheProductionRun.Thisiswherewedotheactualmoleculardynamicsrun.Youwill doa100psrun. Contentsofmd2.in

dynamicsw/PME,DNADrug,10.0cut,50ps &cntrl imin=0,irest=1,ntx=7, ntb=2,pres0=1.0,ntp=1, taup=2, cut=10,ntr=0, ntc=2,ntf=2, tempi=300.0,temp0=300.0, ntt=3,gamma_ln=1.0, nstlim=50000,dt=0.002, ntpr=100,ntwx=500,ntwr=1000 / END

ntp=1mdwithisotropicpositionscaling. ntb=2constantpressure pres0=1referencepressureinbar taup=2.0pressurerelaxationtimeinps Issuethefollowingcommandtorunyourdynamicssimulation.

nohupsanderOimd2.inomd2.outppet.topcpet_md1.rstrpet_md2.rst xpet_md2.mdcrdrefpet_md1.rstinfmd2.info&

Monitortheprogressusingtail 40md2.out.Our100psruntookapproximately11hoursto runona2.7GHzlinuxworkstation.

Analysis
Note:Thesampledatapresentedherewillbedifferentfromyourdataifyoustartedwithcrystal structure.WeusedtheresultfromanAutodockdockingasourstartingcomplexbasedonthe samecrystalstructure. Copy process_mdout.perl toyourworkingprojectdirectory.Usethewhichcommandto determinethelocationofyourperlinterpreter(typewhichperl).OntheSGI,thelocation st (after#!)shouldbe/usr/sbin/perl.Checktomakesurethatthepathintheperlscript(1 linein thescript)matcheswhateverpathwasgivenbytheresultofyourwhichcommand.Makesure thattheprogramisexecutable(usechmod755filename)wherefilename=process_mdout.perl.

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./process_mdout.perlmd1.outmd2.out

TheresultingfilesarereadablebytheGraceprogram(http://plasmagate.weizmann.ac.il/Grace/ )orasspacedelimitedfilesinExcel. Wewilltakealookatsummary.EPTOT(potentialenergyplot).Hereisourplotfora500ps simulation

Thepotentialenergyfluctuatesmildly throughoutthesimulation.Thegeneraltrendistoward lowerenergyafterajumpinenergyduringtherestraineddynamics(waterpreequilibration), whichisagoodsignthatthedynamicsisleadingtowardlowerenergyconformation(s).Plot otherfiles.UseMicrosoftExcelandreadinthefileasaspacedelimitedfile. summary.TEMPgivesthetemperaturefluctuationwithtime. summary.PRESgivesthepressurefluctuationwithtime. Analysisofhydrogenbondsoverthecourseofthetrajectory. Useamolecularviewer(like VMD)toexamineyourstructureforhydrogenbondsbetweenthedrugmoleculeandtheDNA. Pickoutakey hydrogenbondofinteresttoanalyze. Usethefollowinginputfileasatemplate to ptraj.(CAUTION:Youratomnamesforyourdrugmolecule(PET)mightbedifferentthanwhat islistedinthetemplatebelow!)
trajinpet_md2.mdcrd donorDTO2 acceptorPETN1H20 hbonddistance3.5angle120donorDTO2acceptorPETN1H20neighbor2serieshbond

DONOR/ACCEPTORusetospecifythedonoracceptorheavyatoms. DISTANCEusetospecifythecutoffdistanceinangstromsbetweentheheavyatoms participatingintheinteraction. ANGLETheHbondanglecutoff(donorHacceptor)indegrees. SERIES DirectsHbonddatasummarytoSTDOUT.

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ptrajpet.top<hbond.in>pet_hbond.dat

Viewdetailingthehydrogenbondbeinganalyzed.(ImagecreatedusingVMD[11]and Raster3D[12]) Outputfromourhbondanalysis

DONORACCEPTORHACCEPTOR atom#:res@atomatom#:res@atomatom#:res@atom%occupied distanceangle |246:8@O2|803:25@H20801:25@N1|52.802.956(0.16)24.90(13.42)

Thestatisticalanalysisinthepet_hbond.datfilewillbemostimportant.LookforthoseHbonds with high %occupancy(>50%).ThesearethemorestableHbonds.Thehigherthe% occupancythebetter.WhenanalyzingHbonddata,itisbesttoestablishreasonableguidelines forthedistanceandanglecutoffs.ArecentpaperbyChapmanetal.providesanicediscussion ofhydrogenbondcriteria.[13] TheAMBPDBConversionProgram HowtousetheambpdbprogramtoconvertanamberrestartcoordinatefiletoaPDBfile.For example:
ambpdbaatmbresppet_vac.top<pet_avmin.rst>pet_avmin.pdb

TheaatmflaginsuresthatthehydrogenatomnamesconformtoPDBstandardandthebres flaginsuresthattheresiduenamesconformtoPDBstandard. TheRMSplot.Wewillusetheptrajprogram. Contentsofrms.in

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trajinpet_md1.mdcrd trajinpet_md2.mdcrd rmsfirstoutpet_rms.dat:124time1.0

trajin specifiestrajectoryfiletoprocess rmscomputedRMSfittothefirststructureofthefirsttrajectoryreadin. outspecifiesnameofoutputfile :124performrmsdanalysisonresidues1through24only (DNA only).

ptrajpet.top<rms.in xmgracepet_rms.dat

Themodelhasnotequilibratedafter500ps. Thereisaregionofsomestabilitybetween300and 450ps. avg.in

trajinpet_md2.mdcrd300450 center:125 imagecenterfamiliar rmsfirstmassoutav_rms.dat:125 averagepet_avg.rstrest ptrajpet.top<avg.in

Converttherestartfiletoapdbfile.
ambpdbaatmppet.top<pet_avg.rst.1>pet_avg.pdb

Removewaterandionswithanytexteditor. pam_vac.scr 14

sourceleaprc.ff99 sourceleaprc.gaff loadAmberParamspam.frcmod loadAmberPreppam_bcc.prep nap=loadPdbpet_avg.pdb checknap saveAmberParmnappet_avg.toppet_avg.crd Quit tleapsfpam_vac.scr

Minimizeinvacuo(min_vac.in)
oxytocin:invacuominimisationpriortoMD &cntrl imin=1, maxcyc=2500, ncyc=500, ntb=0, igb=0, cut=12 / nohupsanderOimin_vac.inomin_vac.outppet_avg.topcpet_avg.crdr pet_avmin.rst&

UsingX3DNAtoexaminetheDNAstructure. (NOTE:Performthisstepinaseparatedirectory!) TheX3DNAprogramisaveryusefultoolforanalyzingthestereochemicalandother3 dimensionalaspectsofnucleicacids.X3DNAwasdevelopedbyXiangJunLuinWilma OlsonsresearchgroupatRutgersUniversity(seehttp://rutchem.rutgers.edu/~xiangjun/3DNA/ formoreinformationandacopyoftheusermanual).[14]YoumuststartfromaPDBfile. AnalyzeeitheryouraveragestructureorthelastframesavedbyAmber(i.e.therestrtfile convertedtoapdbwithambpdb).

ambpdbbrespfile.top<file_md.rst>file_md.pdb

First,editoutthedrugandallotherHETAMrecordsinatexteditor. Next,youmustaddthe chainID.TodothissplittheDNAchainsintotwoseparatefiles(dna_a.pdbanddna_b.pdb). Usethechangepdbchainid.perlscriptfrom3DDockasfollows

changepdbchainid.perlpdbdna_a.pdbold''newA changepdbchainid.perlpdbdna_b.pdbold''newB

Combinethetwochainsintoonefile(e.g.myfile.pdbordna.pdb)andstartwiththefind_pair utility(usedtoestablishthebasepairinformation).
find_pairmyfile.pdbmyfile.inp

Usetheanalyzeprogramtocarryouttheanalysis.

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analyzemyfile.inp

Younowhaveananalysisofthestructure oftheDNAfromthedrugDNAcomplex. Lookfor thedatainthe*.outfile. Performthesameanalysisinaseparatedirectoryforthecrystal structure(166D.pdb). Comparedifferencesintheminorandmajorgroovewidthsespeciallyin theproximityofwherethedrugbinds(taketheaveragevalueforbasepairs48). Ouraverages revealnodifferencebetweenthemodelandthexraystructure. 10.7 (model PPdistance) vs10.9 (xraystructure PP distance) Questionstoconsider: 1.Theauthors(Nunn,etal.ref#2)claimthattheonlyinteraction(s)theyobserveintheir structureisbetweentheamidiniumNHandtheO4 ofthedeoxyribosesugar.Whathydrogen bondinteractionsdoyouobservefromyourdynamicsrun?

2.Theauthorsnotethatachainofwatermoleculesexistalongthemouthoftheminorgroove justoutsidethebounddrug.Doyouobserveasimilarpatterninthewaterstructureofyour model?(Hint:Usethepet_md.restrtfileconverttoaPDBusingtheambpdbcommandtoview withanyvieweror usemoilviewtoviewthestructure.).

3.PentamidinehasahigherbindingaffinityforthisDNAduplexthandoes goxapentamidine. Maketheappropriatestructuralchangeanddevelopanewprepfileforpentamidineusing antechamber.Performadynamicsrunofthepentamidinecomplexandthe goxapentamidine, eachfor200psduration(i.e.nstlim=100000).Performtheusualanalysis.Howdoes pentamidinecomparewith goxapentamidineintermsofnumberofhydrogenbondswiththe DNA?Howdotheinteractionenergies(Eint =Evdw +Eelec)ofthetwocomplexescompare (Comparetheenergiesoftheaverageinvacuostructuresonly.)?Howdoestheminorgroove spacingcomparebetweenthetwocomplexes(use3DNAormeasurethedistancebetweenP atomsofthephosphatesbypicking4thatbracketthedrugbindingregion.)?
H N H N H O O H N H N H

Pentamidine Bibliography: 1. Case,D.A.,T.E.CheathamIII,T.Darden,H.Gohlke,H.Luo,K.M.Merz,A.Onufriev, C.Simmerling,B.Wang,andR.Woods,TheAmberbiomolecularsimulationprograms. J.Comput.Chem.,2005. 26:p.16681688.

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