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.