纳米技术与生物学研究方法――冷冻电镜

2015.12.8邢福临Nanotechnology&biologyContent•Introduction•DetectionofbiologicalnanoparticlesIntroduction10−3𝑚10−6𝑚10−9𝑚10−10𝑚1mm1μm1nm𝐴𝑜Introduction•Nano-biomaterial•Nanobiologicalapparatus•Nano-biotechnologyinclinicDetectionofbiologicalparticlesprimarystructuresecondarystructuretertiarystructurequaternarystructureDetectionofbiologicalparticlesX-raydiffractionWavelengh~0.01-100𝐴0•Purificationofprotein•Crystallization•Datacollection•Modelingandrevision1953DetectionofbiologicalparticlesX-raydiffraction•Highresolution•Non-destructivetosamples•Nopollution•Informative•Staticconformationonly•HARDtocrystallize!2009Nobleprize:afinestructureofribosomeStructuresof36326biomacromoleculearedeterminedbyX-rayDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)•electronmicroscopeelectronsserveasthesourceofradiation•Electronsareaccelerateddownthemicroscopecolumnatacceleratingvoltagesoftypically80–300kV.•Passingthroughthespecimen•ScatteredelectronsarefocusedbytheelectromagneticlensesofthemicroscopeDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)•Fixation（osmicacid/glutaraldehyde）•dehydration•Staining(heavymetalsalt)•Fltra-thinsectionspreparationAsecondarydamage：theextensivedamagethatresultsfromtheinteractionofelectronswithorganicmatterthelowerelectronapoorsignal-to-noiseratio•electronmicroscopeDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)Mitochondria&endoplasmicreticulumchloroplast•electronmicroscopeDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)•Imagingatliquidnitrogentemperaturesreducestheextentofradiationdamagebyasmuchassixfoldcomparedtoambienttemperatures•Thismeansthatforimagesrecordedatcryogenictemperatures,higherelectrondosescanbeusedtoincreasethesignaltonoiseratio.Howtoincreasethesignal-to-noiseratio?DetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)•averagingimagesofalargenumberofidenticalunits,inanapproachthathasanintellectualconnectiontothewayinwhichscatteringofX-raysbybillionsofmoleculesisaveragedtoobtainstructuralinformationinX-raycrystallographyHowtoobtain3DstructureseachimagetakenatadifferenttiltrelativetothedirectionoftheincidentelectronbeamTheseimagesmaythenbecombinedcomputationally,usingastrategysimilartothatusedincomputerizedaxialtomographyDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)Principleofreconstructionof3DstructurebyFourierinversionprojectionimagesoftheobject,eachwithadifferentorientation,have2DFouriertransformsthatcorrespondtosections(indicatedbyredarrows)throughthe3DFouriertransformoftheoriginalobject.Oncethe3DFouriertransformisbuiltupfromacollectionof2Dimagesspanningacompleterangeoforientations,Fourierinversionenablesrecoveryofthe3Dstructure.DetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)•biologicalspecimensrapidlyfreezedinalayerofglass-likeice•imagedatliquidnitrogenand/orheliumtemperature•averagingmultipleimagescollectedfromfrozen-hydratedspecimensDifferencesbetweenEMandcryo-EMDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)GroELproteincomplexesDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)GroELproteincomplexesatapproximately7Aresolutionusingapproximately28000individualprojectionimagesDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)spliceosomeDetectionofbiologicalparticlesCryo-electronmicroscopy(cryo-EM)references[1]郭强.基于冷冻电镜的原核生物核糖体小亚基组装过程的研究.清华大学,2014.[2].MilneJLS,BorgniaMJ,BartesaghiA,etal.Cryo‐electronmicroscopy–aprimerforthenon‐microscopist.FEBSJournal,2013,280(1):28-45.[3].YanC,HangJ,WanR,etal.Structureofayeastspliceosomeat3.6-angstromresolution.Science,2015,349(6253):1182-1191.[4].HangJ,WanR,YanC,etal.Structuralbasisofpre-mRNAsplicing.Science,2015,349(6253):1191-1198.Thankyou！