CCP4-Tutorial---Session-6

CCP4Tutorial-Session6CCP4Tutorial:Session6-RefinementSeealsotheaccompanyingdocumentgivingbackgroundinformation.ThisisanintroductiontotheproceduresforusingRefmac5torefineacrystalstructure.SeealsothedocumentationforRefmac5andSketcher.Inthefollowinginstructions,whenyouneedtotypesomething,orclickonsomething,itwillbeshowninred.Outputfromtheprogramsortextfromtheinterfaceisgiveningreen.OutlineoftheMethoda.UseRefmactofindanyspecialrestraintsfortheunligandedstructure-forexamplethedisulphidebondsandcis-peptides.Checktheresultsandmakesurethespecialrestraintslistedinthelogfilearecorrect.b.RuntheRefmacprogramtorefinetheunligandedstructureandlookattheresultc.Createthegeometrydescriptionoftheligandd.UseRefmactofindanymorespecialrestraintsfortheligandedforme.RuntheRefmacprogramagaintorefinetheligandedstructureandlookattheresultTheProblemThisexampleistorefinetheproteinRNAseSainitsunligandedandligandedform,forwhichweknow:●Itisasmallprotein;eachmoleculehasonechainof96residues●ThespacegroupisP212121●Therearethreefilesofdata,toresolution2.5Å,1.8Åand1.15Å,eachofwhichcontainsdatafromoneormorecrystalswithorwithoutligands(native,native+2GP,native+3GP)●Forthistutorial,weareinterestedinrefiningthestructureoftheRNAsewithtwosulphateionsandoneligand,guanosine-3*-monophosphate(GMP,or3GPasitiscalledinthemonomerlibrarywewilluse)●Therearetwomoleculesintheasymmetricunit(determinedbycheckingthematthews_coef)●TheREFMACreviewrestraintsprocedurewillsuggestthatthereis:❍Onedisulphidebondintheprotein:cys7-cys96❍Onecis-peptidebondforgly26-pro27Thefinalstructure(solvedbyJosephSevcik:J.Sevcik,Z.Dauter,V.S.Lamzin,K.Wilson,ActaCryst.D52(1996)p327-344)lookslikethis:file:///D|/CCP4-Packages/ccp4-6.0/examples/tutorial/html/refine-tutorial.html(1of17)2007-11-211:30:44CCP4Tutorial-Session6WhatareRestraints?Thereare1749atomsintheasymmetricunit.Ifwedescribeeachatomusingthreepositionalparametersx,y,zandanisotropictemperaturefactorBthereare6996parameters.Intheexperimentaldata(see$DATA/rnase18.mtz)thereare17991reflectionsgivinganobservation-to-parameter-ratioof17991/6996=2.57.Thisisnotenoughtorefineallparametersasindependentvariables.Howeverwehaveagreatdealofinformationaboutthegeometryofmolecules-thebondlengthsandbondanglesetc.Therefinementprogramwillsetuprestraintsbetweenrelatedatomswhichsay,forexample,thatthedistancebetweentwobondedatomsmustbeclosetotheidealbondlength.Refinementprogramsreadlibrariesdescribingtheexpectedgeometry.Thesecontaininformationabouttheidealbondlengths,bondangles,planargroupsetc.forthecommonchemicalmonomerswhicharefoundinmacromolecules(e.g.aminoacidsandnucleicacids).Ifthisinformationisincorrectyouwillnotgetacorrectstructure.Ifyourstructurecontainsanunusualsubstratemoleculeoramodifiedaminoacid,thentheremaybenosuitabledescriptioninthelibrary,andyouwillhavetoprovideoneintherequiredstyle.Thiscanbedifficult;itisessentialyouknowthechemicaldefinitionofyourligand,(e.g.whichatomslieinaplane,whichbondtypeexists,etc).Thisknowledgemustthenbewritteninthecorrectformatfortherefinementprogramtoread.Thiscanalsobechallenging,sowewillmakethegeometrydescriptionfortheligandtoshowhowitisdone.TheDataFilesFilesindirectoryDATA:rnase.pdbTherefinedunligandedproteincoordinatesGMP.pdbIdealisedcoordinatesoftheligandmolecule,guanosine-3*-monophosphaternase-3gp.pdbTherefinedprotein-ligandcoordinatesrnase_bad.pdbAtestsetofcoordinateswitherrorsintroduced.file:///D|/CCP4-Packages/ccp4-6.0/examples/tutorial/html/refine-tutorial.html(2of17)2007-11-211:30:44CCP4Tutorial-Session6rnase18.mtzAnMTZfilecontainingthreesetsofexperimentaldataextendingto1.8Å,labelledFNATSIGFNATF3GPSIGF3GPF2GPSIGF2GPforunligandedprotein,theproteinligandedwithguanosine-3*-monophosphate(3GP)andwithguanosine-2*-monophosphate(2GP),respectivelyrnase115.mtzTheexperimentaldataandcalculatedstructurefactorsandphasesfortheunligandedproteinto1.15Årnase25.mtzAnMTZfilecontainingexperimentaldata(includinganomalous),extendingto2.5Å,forthenativeproteinandthreederivatives(mercury,platinumandiodine),asusedforexperimentalphasingbyMIRandexperimentalphasingbyMADTheoutputfilesindirectoryRESULTS:3GP_mon_lib.cifThemonomerlibrarydescriptionof3GPreview.logThelogfilefromrunningRefmac5toreviewrestraintsrnase18_bad_refmac1.pdbTheoutputfilefromrunningRefmac5toreviewrestraintsrefmac-unliganded.log.logfromRefmac5refinementofunligandedRNAse6a)Checkmodelgeometry,createandreviewspecialrestraintsForthetutorialafilecalled$DATA/rnase_bad.pdbbasbeengenerated,whichhashadsomeerrorsintroduced.TheresidueARGB:63isnowtooclosetoARGA:63.Also,allresidueshavebeenshiftedbyupto0.2Å.WewilluseREFMAC5inreviewrestraintsmode.Theprogramwilllookattheatomcoordinatesanddecidewheretherearedisulphidebonds,cis-peptidesandD-peptides.Itwillalsocalculatethedistancebetweenatomsandiftheyareverycloseitwillassumetheatomsarebondedandwillmakeamakearestrainttosay'thesetwoatomsmuststayclose'.Ofcoursethisisnotalwaysright.Itwillalsoaddanyabsentatoms-ifaresiduedoesnothavetherightatomsitwillmakethem.REFMAC5willhelpyoubyfindingthedisulphidebondsetc.automaticallybutyouMUSTchecktha