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简介：Micro-electrondiffraction(MicroED)isanemergingtechniquetousecryo-electronmicroscopetostudythecrystalstructuresofmacromoleculefromitsmicro-/nano-crystals,whicharenotsuitableforconventionalX-raycrystallography.However,thistechniquehasbeenpreventedforitswideapplicationbythelimitedavailabilityofproducinggoodmicro-/nano-crystalsandtheinappropriatetransferofcrystals.Here,wedevelopedacompleteworkflowtopreparesuitablecrystalsefficientlyforMicroEDexperiment.Thisworkflowincludesinsituon-gridcrystallization,single-sideblotting,cryo-focusionbeam(cryo-FIB)fabrication,andcryo-electrondiffractionofcrystalcryo-lamella.ThisworkflowenablesustoapplyMicroEDtostudymanysmallmacromolecularcrystalswiththesizeof2-10μm,whichistoolargeforMicroEDbutquitesmallforconventionalX-raycrystallography.Wehaveappliedthismethodtosolve2.5Acrystalstructureoflysozymefromitsmicro-crystalwithinthesizeof10×10×10μm^3.OurworkwillgreatlyexpandtheavailabilityspaceofcrystalssuitableforMicroEDandfillupthegapbetweenMicroEDandX-raycrystallography.

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简介：Theplasmamembrane(PM)isacomplexenvironmentconsistingof>700speciesoflipidsandmanydifferenttypesofmembrane-associatingproteins.Theselipidsandmembraneproteinsaredistributedheterogeneouslyintonanometer-sizeddomains,callednanoclusters.ThelateralspatialsegregationinthePMgivesrisetodifferentcurvatureandlipidcomposition,whichdeterminestheefficiencyofeffectorbindingandsignaltransmission.Here,wedescribeanelectronmicroscopy(EM)-spatialmappingtechniquetoquantifytheextentofnanoclustersformationinthePM.Thenano-assembliesinthePMarequantifiedviaexpressingtheGFP-taggedproteinsorlipid-bindingdomainsinthecells,whicharethenimmunolabeledwiththegoldnanoparticlespre-coupledtotheanti-GFPantibody.ThegoldnanoparticlesarevisualizedviathetransmissionEMathighmagnification.ThestatisticalanalysisoftheRipley'sK-functioncalculatesthespatialdistributionofthegoldnanoparticles.Importantspatialparameters,suchastheextentofnanoclustering,theclusteredfraction,thenumberofproteinspercluster,theoptimalsizeofananocluster,andthenumberofproteinslocalizedtothePM,canbecalculated.Furtherdetailedaggregationpattern,suchasthepopulationsofmonomers,dimers,trimers,andhigherorderedoligomers,canalsobeextractedfromthespatialanalysis.TheEM-bivariateanalysisquantifiestheextentofco-localizationbetweentwodifferentcomponentsinthePMandprovideskeyinformationontheprotein-proteinandtheprotein-lipidinteractionsoveralong-distancescalefrom8to240nm.

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