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1、ArticleProteinScienceDOI10.1002/pro.3048Crucialstepsinthestructuredeterminationofacoronavirusspikeglycoproteinusingcryo-electronmicroscopy1*2*3*1AlexandraWalls,M.AlejandraTortorici,Berend-JanBosch,BrandonFrenz,121*PeterJ.M.Rottier,FrankDiMaio,FelixReyandDavidV

2、eesler.1DepartmentofBiochemistry,UniversityofWashington,Seattle,Washington98195,USA.2InstitutPasteur,DépartementdeVirologie,UnitédeVirologieStructurale,Paris,France.CNRSUMR3569Virologie,Paris,France.3VirologyDivision,DepartmentofInfectiousDiseasesandImmunology

3、,FacultyofVeterinaryMedicine,UtrechtUniversity,Utrecht,TheNetherlands.*Theseauthorscontributedequallytothiswork.Thisarticlehasbeenacceptedforpublicationandundergonefullpeerreviewbuthasnotbeenthroughthecopyediting,typesetting,paginationandproofreadingprocesswhi

4、chmayleadtodifferencesbetweenthisversionandtheVersionofRecord.Pleasecitethisarticleasdoi:10.1002/pro.3048?2016TheProteinSocietyReceived:Aug31,2016;Revised:Sep21,2016;Accepted:Sep22,2016Thisarticleisprotectedbycopyright.Allrightsreserved.ProteinSciencePage2of22

5、Thetremendouspandemicpotentialofcoronaviruseswasdemonstratedtwiceinthelast15yearsbytwoglobaloutbreaksofdeadlypneumonia.EntryofcoronavirusesintocellsismediatedbythetransmembranespikeglycoproteinS,whichformsatrimercarryingreceptor-bindingandmembranefusionfunctio

6、ns.Despitetheirbiomedicalimportance,coronavirusSglycoproteinshaveprovendifficulttargetsforstructuralcharacterization,precludinghigh-resolutionstudiesofthebiologicallyrelevanttrimer.Recenttechnologicaldevelopmentsinsingleparticlecryo-electronmicroscopyallowedus

7、todeterminethefirststructureofacoronavirusSglycoproteintrimerwhichprovidedaframeworktounderstandthemechanismsofviralentryandsuggestedpotentialinhibitionstrategiesforthisfamilyofviruses.Here,wedescribethekeyfactorsthatenabledthisbreakthrough.Keywords:Coronaviru

8、sspikeprotein,cryo-electronmicroscopy,rationalvaccinedesign,Rosetta,RelionBroadAudienceStatement:Therecentemergenceofhighlypathogeniccoronavirusesandthepotentialforfutureoutbreaksh