•INSIGHT•
SCIENCECHINA
LifeSciences
November2015Vol.58No.11:1154–1156doi:10.1007/s11427-015-4942-
0 Nolonger‘blob-ology’:Cryo-EMisgettingintomoleculardetails ZHUHongTao&ZHUPing* NationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,ChinaReceivedSeptember17,2005;eptedSeptember26,2015;publishedonlineOctober12,2015 Citation: ZhuHT,ZhuP.Nolonger‘blob-ology’:Cryo-EMisgettingintomoleculardetails.SciChinaLifeSci,2015,58:1154–1156,doi:10.1007/s11427-015-4942-
0 Thestructuralinformationoffunctionallyimportantmacromolecularassembliesisakeytomolecularbiologyandisofgreatinteresttostructuralbiologists.Cryo-electronmicroscopy(cryo-EM),particularlythesingleparticleanalysis(SPA),hasbeenregardedasoneofthethreeprimarytechniquesforstructuredetermination,togetherwithX-raycrystallographyandnuclearicresonance(NMR).Forsingleparticlecryo-EM,atinyamount(~3L)ofpurifiedbiologicalsamplesataconcentrationaround0.12mgmL1areplacedontoanEMgrid,blottedwithafilterpaperandrapidlyfrozenintoathinlayerofvitreousice.TheEMgridwithfrozensamplesisthentransferredtoatransmissionelectronmicroscopeforimaging.Thousandsofmicrographsaretakenandmillionsofimagesofindividualmacromolecularparticlesarepicked,putationallyalignedandaveragedtoacquireathree-dimensionalstructure. Althoughthetechniquesandprincipleofcryo-EMreconstructionhadbeendevelopedforseveraldecades,theresolutionofstructuresdeterminedbycryo-EMhaslongbeenlimitedtonanometertosub-nanometerrange.Thelow-tomedium-resolutionstructure,i.e.,3-Ddensitymap,generatedbycryo-EMreconstruction,althoughveryhelpfulinunderstandingthegeneralassemblyofthemacromolecules,oftenappearsasa“blob”withoutdetailedfeatures.Consequently,cryo-EMtechniquehadbeentermedas‘blob-ology’formanyyears. However,thissituationhasbeenchangedwithanumberofbreakthroughsmadeincryo-EMfieldinthepasttwoyears.In2013,Liaoetal.[1]solvedthestructureof *Correspondingauthor(email:zhup@) TRPV1,anionchannelmembraneprotein,at3.4Åresolutionbysingleparticleanalysis.Thisisthoughttobegroundbreakingbecauseitopensadoorformembraneproteinstructuredeterminationwithoutcrystallization,whichis thebottleneckofX-raycrystallography—thecurrently dominanttechniqueformembraneproteinstructuredetermination.Atthesametime,significantprogresshadalsobeenmadeonthecryo-EMstructuredeterminationofothermacromolecularassembliesormulti-subunitplexes.Forexample,thestructureofthelargesubunitofayeastmitochondrialribosomehadbeensolvedbysingleparticleanalysisat3.2Åresolution[2],whichcanonlybeachievedforhighlysymmetricalicosahedralvirusesbycryo-EMpreviously.Attheseresolutions,atomicmodelsofmacromolecularassembliesormulti-subunitplexescanbebuiltdenovo,i.e.,solelybasedonthecryo-EMdensitymapswithoutthehelpofatomicstructuresofthesub-unitsdeterminedbyeitherX-raycrystallographyorNMRtechnique. Thistermed“resolutionrevolution”incryo-EMisaconsequenceofseriesofadvancesmaderecently,bothinhardwareequipmentsanddataprocessingprocedures.Thefirst,andprobablythemostimportantone,isbinationofrecentlydevelopeddirectelectrondetectordevice(DDD)withstate-of-the-artcryo-electronmicroscope.Comparedwithcharge-coupleddetectors(CCD)whichareinstalledinmostofthehigh-endelectronmicroscopes,thedirectelectrondetectorhasanoteworthyimprovementinthedetectivequantumefficiency(DQE).TherecordedEMimagewithDDDpresentsaremarkablybettersignaltonoiseratio(SNR)thanthatwithCCD.Thismakesthesub- ©TheAuthor(s)2015.Thisarticleispublishedwithopenessat ZhuHT,etal.SciChinaLifeSciNovember(2015)Vol.58No.11 1155 sequentimageprocessingmuchmoreurateandefficient.Inaddition,thenewlyadopteddirectelectronrecordingdevicemakesitpossibletocorrectthebeam-inducedsamplemovement.Exposedtochargedelectronbeamsintheelectronmicroscope,biologicalsamplesburiedinvitreousicearesubjecttobeam-inducedmovementinstochasticdirections,whichmakesrecordedEMmicrographsshakywithblurrydetailsandhamperstheachievementofatomicstructuresbysubsequentimageanalysis.Byusingthenewlyinstalleddirectelectrondetectors,dose-fractionatedimagestackscanberecordedinmoviemodeandthebeam-inducedspecimenmovementscanputationallycalculatedandcorrected,whichsignificantlyimprovetheimagequalityandgreatlyhelpthesubsequentstructuredeterminationtoadvanceintoanatomicresolutionlevel[3,4]. Anothercriticaldevelopmentattributedtothecryo-EM“resolutionrevolution”istheimprovementofimageprocessingprocedures.Theintrinsicstructuralheterogeneitywithinsamples,e.g.,theconformationalchangeofbiologicalsamplesindifferentstates,makesitdifficultforresearcherstoaligntheimagespreciselyandtoresolvetheatomicdetailsofmacromoleculesbycryo-EM.Themethodsofmaximum-likelihoodbasedimagealignmentandheterogeneoussampleclassificationhavegreatlyimprovedtheresolutionofstructuredeterminationinsingleparticleanalysis.Asoftwarepackage,RELION[5],whichintegratesthesemethodsisnowwidelyusedincryo-EMfield. Withthehelpofthesedramatictechnicaladvances,numerouscryo-EMstructuresatatomicresolutionlevelhavebeenachievedforarangeofspecimens,fromsmallmembraneproteinstomulti-subunitsplexes.Forsmallmembraneproteins,thestructureofhumanγ-secretase,anasymmetricmembraneproteinwithmolecularweightof170kD,hasbeenrecentlydeterminedat3.4Åresolution[6].Formulti-subunitsplexes,aseriesoflong-awaitingstructureshavebeensolvedatnear-atomicresolution,forexample,thestructureofplex,aplex,hasbeendeterminedatthe4.0Åresolution[7]andthestructureofayeastspliceosome,a37plex,hasbeenresolvedat3.6Åresolution[8].Havingbeencontroversialforseveraldecades,thestructureof30nmchromatinfiber,anponentofnucleus,wasalsosolvedbycryo-EMat11ÅresolutionwithCCDimages[9].Withouttherecentadvancesincryo-EMtechnique,todeterminethestructuresofthesehuge,sometimesflexible,plexeswouldbehighlychallenging,ifnotimpossible. Currently,theresolutionrecordofstructuredeterminationbysingleparticlecryo-EMhasbeensetto2.2Å[10],whichisnowbeginningtorivalX-raycrystallography.Nolongera‘‘blob-ology’’,cryo-EMisonthewaytoplayoneofthemajorrolesinstructuredeterminationofplexesatatomicresolution. Nevertheless,variouseffortsarestillneededtomakecryo-EMapowerful,convenientandattractivetoolfor structuredetermination.(i)Improvestructuredeterminingresolutiontoahigher level.Newgenerationsofelectrondetectorswiththinnerchipandfasterreadoutsarebeingdeveloped,whichmayyieldabetterDQEclosetothephysicallimit.Moreefficientmotioncorrectionalgorithms,newtypesofEMgridorsupportingfilmsarealsobeingdevelopedtofurtherreduceorpletelythebeam-inducedsamplemovement.Abetterresolution,e.g.,1.5Å,willmakecryo-EMusefulforstructure-baseddrugorinedesign,andattractivetothepharmaceuticalindustry. (ii)Workwithmoreflexibleandstructurallyheterogeneoussamples.Theintrinsicflexibilityordynamiccharacteristicsofbiologicalsamplespostagreatchallengetothestructuredeterminationatatomicresolution.Betterimageclassificationmethodsarebeingdevelopedtodealwithbiologicalsampleswithlargeramountsofstructuralheterogeneity.Itwouldbeindispensibleifcryo-EMcouldrevealthestructuredynamicsofplexesthroughextensiveclassificationsofhundredsofmillionsofparticlesindifferentstatus. (iii)Breakthebottleneckofbiochemicalsamplepreparation.Whilehigh-throughputcryo-EMstructuredeterminationisontheway,thebiochemicalsamplepreparationesthemainbottleneck.Anefficientmethodtoproducesufficientsamplesforcryo-EMstructuredeterminationisclearlyineagerneed.Withrelativelylesssamplerequiredforcryo-EM,extractingendogenousmacromolecularassembliesfromcellsortissuesandsolvingtheirstructuresinnativestatuswouldefeasible. (iv)Makecryo-EMlessexpensiveandmoreessible.Currently,theestablishmentandmaintenanceofastate-of-the-artcryo-EMfacilityishighlycostlyandthewholeprocessofcryo-EMstructuredeterminationisquiteexperiencedemanding.Thewholefieldwouldbenefitgreatlyfromimprovedessibilityofthecryo-EMequipmentandtechnique. Withnumeroustechnologyadvancesontheroad,cryo-EMisnowrevolutionizingthestructuralbiologyandpavingthewayfora“goldenera”. 1LiaoM,CaoE,JuliusD,ChengY.StructureoftheTRPV1ionchanneldeterminedbyelectroncryo-microscopy.Nature,2013,504:107–112 2AmuntsA,BrownA,BaiXC,LlacerJL,HussainT,EmsleyP,LongF,MurshudovG,ScheresSH,RamakrishnanV.Structureoftheyeastmitochondriallargeribosomalsubunit.Science,2014,343:1485–1489 3LiX,MooneyP,ZhengS,BoothCR,BraunfeldMB,GubbensS,AgardDA,ChengY.Electroncountingandbeam-inducedmotioncorrectionenablenear-atomic-resolutionsingle-particlecryo-EM.NatMethods,2013,10:584–590 4BaiXC,FernandezIS,McMullanG,ScheresSH.Ribosomestructurestonear-atomicresolutionfromthirtythousandcryo-EMparticles.eLife,2013,2:e00461 5ScheresSH.RELION:implementationofaBayesianapproachto 1156 ZhuHT,etal.SciChinaLifeSciNovember(2015)Vol.58No.11 cryo-EMstructuredetermination.JStructBiol,2012,180:5195306BaiXC,YanC,YangG,LuP,MaD,SunL,ZhouR,ScheresSH, ShiY.Anatomicstructureofhumangamma-secretase.Nature,2015,525:212–2177UrnaviciusL,ZhangK,DiamantAG,MotzC,SchlagerMA,YuM,PatelNA,RobinsonCV,CarterAP.Thestructureoftheplexanditsinteractionwithdynein.Science,2015,347:1441–14468YanC,HangJ,WanR,HuangM,WongCC,ShiY.Structureofa yeastspliceosomeat3.6-angstromresolution.Science,2015,349:1182–11919SongF,ChenP,SunD,WangM,DongL,LiangD,XuRM,ZhuP,LiG.Cryo-EMstudyofthechromatinfiberrevealsadoublehelixtwistedbytetranucleosomalunits.Science,2014,344:376–38010BartesaghiA,MerkA,BanerjeeS,MatthiesD,WuX,MilneJL,SubramaniamS.2.2Åresolutioncryo-EMstructureofbeta-galactosidaseplexwithacell-permeantinhibitor.Science,2015,348:1147–1151 OpenessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsanyuse,distribution,andreproductioninanymedium,providedtheoriginalauthor(s)andsourcearecredited.
0 Nolonger‘blob-ology’:Cryo-EMisgettingintomoleculardetails ZHUHongTao&ZHUPing* NationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,ChinaReceivedSeptember17,2005;eptedSeptember26,2015;publishedonlineOctober12,2015 Citation: ZhuHT,ZhuP.Nolonger‘blob-ology’:Cryo-EMisgettingintomoleculardetails.SciChinaLifeSci,2015,58:1154–1156,doi:10.1007/s11427-015-4942-
0 Thestructuralinformationoffunctionallyimportantmacromolecularassembliesisakeytomolecularbiologyandisofgreatinteresttostructuralbiologists.Cryo-electronmicroscopy(cryo-EM),particularlythesingleparticleanalysis(SPA),hasbeenregardedasoneofthethreeprimarytechniquesforstructuredetermination,togetherwithX-raycrystallographyandnuclearicresonance(NMR).Forsingleparticlecryo-EM,atinyamount(~3L)ofpurifiedbiologicalsamplesataconcentrationaround0.12mgmL1areplacedontoanEMgrid,blottedwithafilterpaperandrapidlyfrozenintoathinlayerofvitreousice.TheEMgridwithfrozensamplesisthentransferredtoatransmissionelectronmicroscopeforimaging.Thousandsofmicrographsaretakenandmillionsofimagesofindividualmacromolecularparticlesarepicked,putationallyalignedandaveragedtoacquireathree-dimensionalstructure. Althoughthetechniquesandprincipleofcryo-EMreconstructionhadbeendevelopedforseveraldecades,theresolutionofstructuresdeterminedbycryo-EMhaslongbeenlimitedtonanometertosub-nanometerrange.Thelow-tomedium-resolutionstructure,i.e.,3-Ddensitymap,generatedbycryo-EMreconstruction,althoughveryhelpfulinunderstandingthegeneralassemblyofthemacromolecules,oftenappearsasa“blob”withoutdetailedfeatures.Consequently,cryo-EMtechniquehadbeentermedas‘blob-ology’formanyyears. However,thissituationhasbeenchangedwithanumberofbreakthroughsmadeincryo-EMfieldinthepasttwoyears.In2013,Liaoetal.[1]solvedthestructureof *Correspondingauthor(email:zhup@) TRPV1,anionchannelmembraneprotein,at3.4Åresolutionbysingleparticleanalysis.Thisisthoughttobegroundbreakingbecauseitopensadoorformembraneproteinstructuredeterminationwithoutcrystallization,whichis thebottleneckofX-raycrystallography—thecurrently dominanttechniqueformembraneproteinstructuredetermination.Atthesametime,significantprogresshadalsobeenmadeonthecryo-EMstructuredeterminationofothermacromolecularassembliesormulti-subunitplexes.Forexample,thestructureofthelargesubunitofayeastmitochondrialribosomehadbeensolvedbysingleparticleanalysisat3.2Åresolution[2],whichcanonlybeachievedforhighlysymmetricalicosahedralvirusesbycryo-EMpreviously.Attheseresolutions,atomicmodelsofmacromolecularassembliesormulti-subunitplexescanbebuiltdenovo,i.e.,solelybasedonthecryo-EMdensitymapswithoutthehelpofatomicstructuresofthesub-unitsdeterminedbyeitherX-raycrystallographyorNMRtechnique. Thistermed“resolutionrevolution”incryo-EMisaconsequenceofseriesofadvancesmaderecently,bothinhardwareequipmentsanddataprocessingprocedures.Thefirst,andprobablythemostimportantone,isbinationofrecentlydevelopeddirectelectrondetectordevice(DDD)withstate-of-the-artcryo-electronmicroscope.Comparedwithcharge-coupleddetectors(CCD)whichareinstalledinmostofthehigh-endelectronmicroscopes,thedirectelectrondetectorhasanoteworthyimprovementinthedetectivequantumefficiency(DQE).TherecordedEMimagewithDDDpresentsaremarkablybettersignaltonoiseratio(SNR)thanthatwithCCD.Thismakesthesub- ©TheAuthor(s)2015.Thisarticleispublishedwithopenessat ZhuHT,etal.SciChinaLifeSciNovember(2015)Vol.58No.11 1155 sequentimageprocessingmuchmoreurateandefficient.Inaddition,thenewlyadopteddirectelectronrecordingdevicemakesitpossibletocorrectthebeam-inducedsamplemovement.Exposedtochargedelectronbeamsintheelectronmicroscope,biologicalsamplesburiedinvitreousicearesubjecttobeam-inducedmovementinstochasticdirections,whichmakesrecordedEMmicrographsshakywithblurrydetailsandhamperstheachievementofatomicstructuresbysubsequentimageanalysis.Byusingthenewlyinstalleddirectelectrondetectors,dose-fractionatedimagestackscanberecordedinmoviemodeandthebeam-inducedspecimenmovementscanputationallycalculatedandcorrected,whichsignificantlyimprovetheimagequalityandgreatlyhelpthesubsequentstructuredeterminationtoadvanceintoanatomicresolutionlevel[3,4]. Anothercriticaldevelopmentattributedtothecryo-EM“resolutionrevolution”istheimprovementofimageprocessingprocedures.Theintrinsicstructuralheterogeneitywithinsamples,e.g.,theconformationalchangeofbiologicalsamplesindifferentstates,makesitdifficultforresearcherstoaligntheimagespreciselyandtoresolvetheatomicdetailsofmacromoleculesbycryo-EM.Themethodsofmaximum-likelihoodbasedimagealignmentandheterogeneoussampleclassificationhavegreatlyimprovedtheresolutionofstructuredeterminationinsingleparticleanalysis.Asoftwarepackage,RELION[5],whichintegratesthesemethodsisnowwidelyusedincryo-EMfield. Withthehelpofthesedramatictechnicaladvances,numerouscryo-EMstructuresatatomicresolutionlevelhavebeenachievedforarangeofspecimens,fromsmallmembraneproteinstomulti-subunitsplexes.Forsmallmembraneproteins,thestructureofhumanγ-secretase,anasymmetricmembraneproteinwithmolecularweightof170kD,hasbeenrecentlydeterminedat3.4Åresolution[6].Formulti-subunitsplexes,aseriesoflong-awaitingstructureshavebeensolvedatnear-atomicresolution,forexample,thestructureofplex,aplex,hasbeendeterminedatthe4.0Åresolution[7]andthestructureofayeastspliceosome,a37plex,hasbeenresolvedat3.6Åresolution[8].Havingbeencontroversialforseveraldecades,thestructureof30nmchromatinfiber,anponentofnucleus,wasalsosolvedbycryo-EMat11ÅresolutionwithCCDimages[9].Withouttherecentadvancesincryo-EMtechnique,todeterminethestructuresofthesehuge,sometimesflexible,plexeswouldbehighlychallenging,ifnotimpossible. Currently,theresolutionrecordofstructuredeterminationbysingleparticlecryo-EMhasbeensetto2.2Å[10],whichisnowbeginningtorivalX-raycrystallography.Nolongera‘‘blob-ology’’,cryo-EMisonthewaytoplayoneofthemajorrolesinstructuredeterminationofplexesatatomicresolution. Nevertheless,variouseffortsarestillneededtomakecryo-EMapowerful,convenientandattractivetoolfor structuredetermination.(i)Improvestructuredeterminingresolutiontoahigher level.Newgenerationsofelectrondetectorswiththinnerchipandfasterreadoutsarebeingdeveloped,whichmayyieldabetterDQEclosetothephysicallimit.Moreefficientmotioncorrectionalgorithms,newtypesofEMgridorsupportingfilmsarealsobeingdevelopedtofurtherreduceorpletelythebeam-inducedsamplemovement.Abetterresolution,e.g.,1.5Å,willmakecryo-EMusefulforstructure-baseddrugorinedesign,andattractivetothepharmaceuticalindustry. (ii)Workwithmoreflexibleandstructurallyheterogeneoussamples.Theintrinsicflexibilityordynamiccharacteristicsofbiologicalsamplespostagreatchallengetothestructuredeterminationatatomicresolution.Betterimageclassificationmethodsarebeingdevelopedtodealwithbiologicalsampleswithlargeramountsofstructuralheterogeneity.Itwouldbeindispensibleifcryo-EMcouldrevealthestructuredynamicsofplexesthroughextensiveclassificationsofhundredsofmillionsofparticlesindifferentstatus. (iii)Breakthebottleneckofbiochemicalsamplepreparation.Whilehigh-throughputcryo-EMstructuredeterminationisontheway,thebiochemicalsamplepreparationesthemainbottleneck.Anefficientmethodtoproducesufficientsamplesforcryo-EMstructuredeterminationisclearlyineagerneed.Withrelativelylesssamplerequiredforcryo-EM,extractingendogenousmacromolecularassembliesfromcellsortissuesandsolvingtheirstructuresinnativestatuswouldefeasible. (iv)Makecryo-EMlessexpensiveandmoreessible.Currently,theestablishmentandmaintenanceofastate-of-the-artcryo-EMfacilityishighlycostlyandthewholeprocessofcryo-EMstructuredeterminationisquiteexperiencedemanding.Thewholefieldwouldbenefitgreatlyfromimprovedessibilityofthecryo-EMequipmentandtechnique. Withnumeroustechnologyadvancesontheroad,cryo-EMisnowrevolutionizingthestructuralbiologyandpavingthewayfora“goldenera”. 1LiaoM,CaoE,JuliusD,ChengY.StructureoftheTRPV1ionchanneldeterminedbyelectroncryo-microscopy.Nature,2013,504:107–112 2AmuntsA,BrownA,BaiXC,LlacerJL,HussainT,EmsleyP,LongF,MurshudovG,ScheresSH,RamakrishnanV.Structureoftheyeastmitochondriallargeribosomalsubunit.Science,2014,343:1485–1489 3LiX,MooneyP,ZhengS,BoothCR,BraunfeldMB,GubbensS,AgardDA,ChengY.Electroncountingandbeam-inducedmotioncorrectionenablenear-atomic-resolutionsingle-particlecryo-EM.NatMethods,2013,10:584–590 4BaiXC,FernandezIS,McMullanG,ScheresSH.Ribosomestructurestonear-atomicresolutionfromthirtythousandcryo-EMparticles.eLife,2013,2:e00461 5ScheresSH.RELION:implementationofaBayesianapproachto 1156 ZhuHT,etal.SciChinaLifeSciNovember(2015)Vol.58No.11 cryo-EMstructuredetermination.JStructBiol,2012,180:5195306BaiXC,YanC,YangG,LuP,MaD,SunL,ZhouR,ScheresSH, ShiY.Anatomicstructureofhumangamma-secretase.Nature,2015,525:212–2177UrnaviciusL,ZhangK,DiamantAG,MotzC,SchlagerMA,YuM,PatelNA,RobinsonCV,CarterAP.Thestructureoftheplexanditsinteractionwithdynein.Science,2015,347:1441–14468YanC,HangJ,WanR,HuangM,WongCC,ShiY.Structureofa yeastspliceosomeat3.6-angstromresolution.Science,2015,349:1182–11919SongF,ChenP,SunD,WangM,DongL,LiangD,XuRM,ZhuP,LiG.Cryo-EMstudyofthechromatinfiberrevealsadoublehelixtwistedbytetranucleosomalunits.Science,2014,344:376–38010BartesaghiA,MerkA,BanerjeeS,MatthiesD,WuX,MilneJL,SubramaniamS.2.2Åresolutioncryo-EMstructureofbeta-galactosidaseplexwithacell-permeantinhibitor.Science,2015,348:1147–1151 OpenessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsanyuse,distribution,andreproductioninanymedium,providedtheoriginalauthor(s)andsourcearecredited.
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