CCP4-Tutorial

CCP4Tutorial:MolecularReplacementandMrBUMPThistutorialdemonstrateshowtousetheMrBUMPprogramtocarryoutmuchoftheworkinvolvedinthemolecularreplacement(MR)processautomatically.Firstly,however,itisimportantforthestudentstogainsomeunderstandingoftheunderlyingstepsandprogramsinvolved.WewillstartwithanexampleofhowtomanuallypreparethedataandasetofsearchmodelsforMR.WewillthenperformtheMRusingbothMolrepandPhaser.Finally,willgetMrBUMPtoperformthewholeprocessautomatically.WewillsolvethehypFstructurebymolecularreplacement,usingseveralprogramsandapproaches.OtherMRexamplescanbefoundattheendofthistutorial.WhenthistutorialisobtainedaspartoftheCCP4distribution,$MR_TUTORIALcorrespondsto$CCP4/examples/mr_tutorial_2006.ItisagoodideatosetupanewprojectinCCP4ithatcorrespondstothisdirectory.CheckingthedataTargetistheacylphosphatase-likedomainofhydrogenasematurationfactorHypFfromE.coli,seeRosanoetal,JMB,321,785(2002).HypF-ACPsulphateandphosphatecomplexesdepositedas1gxtand1gxurespectively.ThisproteinhasaHgderivative.Youmayhaveprocessedthisdatainaprecedingtutorial.Wehavepreparedareflectionfileforyouincludingthedatafrom1gxu,1gxt,theHgderivative,andsomeexperimentalphasesbasedontheHgsites.ThereisnativedatainH32to1.3Aresolution.Thetargethas91residuesandaMatthewscalculationstronglysuggestsonlyonemoleculeintheasymmetricunit.WefirstuseSfchecktocheckafewthingsaboutthedata:1.SelecttheProgramListmoduleandopenthesfchecktaskwindow.2.Enteratitle.3.Un-checkRunRampagetoanalysestructuregeometryandRunProchecktoanalysestructuregeometry(wedonotyethaveanycoordinates)4.SelectRunSfchecktoanalyseexperimentaldataonly5.EnterMTZin$MR_TUTORIAL/data/hypF/hypF-1gxu-1gxt-HG_scaleit1.mtzandselectthelabelsFFP1gxu,SIGFSIGFP1gxuandFreeFREE6.CheckthatasuitablefilenamehasbeengeneratedforSfcheckOutputPS7.Keepalldefaults,andclickRun-RunNow.Sfcheckproducesapostscriptfilewithsomeusefulthings(seeunderViewFilesfromJob):Anisotropyofdata(itisnotveryanisotropic)OverallBfromWilsonplotof21.9A**2Pseudo-translationnotdetected(fromanalysisofthenativePatteronmap)AlsocheckthelogfileViewFilesfromJobthenViewLogFile:Thisincludestheresultsoftheatwinningtest:PerfecttwinningtestI^2/I^2:2.0573Avalueof2.0indicatesuntwinneddata,whereasperfectlytwinneddatawouldhaveasecondmomentof1.5ChoiceofsearchmodelsThetargetisanacylphosphatase-likedomain.AsearchofthePDBrevealstwoacylphosphataseswithasequenceidentitytothetargetofabout31%-1v3zand1w2i.Eachhastwochainsintheasymmetricunit,eitherofwhichcouldbeusedasthebasisofasearchmodel.NormallyyouwouldusesomethinglikeChainsawatthispointtoprepareasearchmodelfromthetemplate.Asanexercise,wearegoingtotryMRstraightaway.WewillreturntoChainsawlaterbeforerunningPhaser.NotesonSequenceAlignmentTherearemanywaysofapproachingthis,andthedifferenttoolswillgiveslightlydifferentassessments.Thesequenceidentitydependsonthedefinitionsused(i.e.treatmentofgapsandalignmentlength),thespecificalignmenttechnique,andwhetherbitshavebeenchoppedoutofthemodel.MolrepRun1WewillusechainBof1v3zasthesearchmodel(file$MR_TUTORIAL/data/hypF/1v3z_B.pdb).1.SelecttheMolecularReplacementmoduleandopentheRunMolrep-autoMRtaskwindow.2.Enteratitle.3.Domolecularreplacementperformingrotationandtranslationfunctionshouldbealreadyselected.4.EntertheMTZfile$MR_TUTORIAL/data/hypF/hypF-1gxu-1gxt-HG_scaleit1.mtzandselectthelabelsFPFP1gxuandSIGFPSIGFP1gxu5.EnterthePDBfile$MR_TUTORIAL/data/hypF/1v3z_B.pdb6.(Optional)Youcanuseanupperresolutioncutoffof3Atospeedupthecalculation,seefolderExperimentalData.7.Keepalldefaults,andclickRun-RunNow.Whenthejobhasfinished,lookatthelogfile(ViewFilesfromJob-ViewLogFile).Notethefollowing:Molrepautomaticallyestimates:INFO:expectednumberofmonomers:1Vmol:61.4%whichiscorrect.Theestimatemaybeunreliablewhentherearemanymonomersintheasymmetricunit,inwhichcaseitcanbesetexplicitlywiththekeywordNMON(seefolderSearchParametersintheMolrepGUI).Molrepcheckswhetherornotananisotropycorrectionisnecessary:INFO:AnisotropicywillnotbeusedThefirsttableisalistofpeaksoftheCrossRotationFunction(CRF),sortedaccordingtotheirheights.ThesecondtableshowsthebestTranslationFunction(TF)foreachoftheCRFpeaks(scoredaccordingtothecorrelationcoefficient*PKmax).OtherTFsolutionscanbeviewedinthefileViewFilesfromJob-OutputFiles...proj_dir_job_no_molrep.docThefinaltablegivesalistofsolutions,sortedaccordingtothescore.Molrepreports:INFO:contrastisgoodenough.Stopthisrunbasedonacontrastof3.29(theprecisevalueyougetwilldependonMolrepversion,resolutionlimitsused,etc.)Run2Infact,wecanmakeuseofourknowledgeofthetarget,andthiswilloftenimprovethesolution.Thesearchmodelhasamoderatelylowsequenceidentitywiththetargetandthereforethemajorityofthesidechainsareincorrect.Molrepcanmakeuseofthetargetsequencetoimprovethesearchmodel.1.Selectthepreviousjob,andclickReRunJob2.Mostoftheparametersshouldbesetcorrectly,butyoushouldchangethetitle,andthenameoftheCoordsoutfile,sothatitisdifferentfromthefirstjob.3.Thistime,selectUsesequenceintheprotocolsection.Afolderwillopenbelowwhereyoucanspecifythenameofthetargetsequencefile$MR_TUTORIAL/data/hypF/hypF_Nd