2021-03-10RELRCSBRCSBRCSB2020-05-102021-01-202021-01-202021-03-10National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)CA-13202United StatesCryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Upright conformationHerrmann THarrison SCJenni SHerrmann TTorres RSalgado ENBerciu CStoddard DNicastro DJenni SHarrison SCFunctional refolding of the penetration protein on a non-enveloped virus.NatureUK590666670202133442061doi:10.1038/s41586-020-03124-41476-46870006NATUASEMD-21955associated EM volumeCryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Spike conformation6wxeFULLOVERLAPRotavirus VP4, VP6, VP7 assembly in the spike conformationRotavirus VP4, VP6, VP7 assembly in the spike conformation0123Obtained from wild-type recoated rhesus rotavirus particles (wt rcTLPs)Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])1.7Outer capsid protein VP4Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])RVA/Monkey/United States/RRV/1975/G3P5B[3]0.086655585999999993Spodoptera frugiperdaLEVOMASLIYRQLLTNSYTVDLSDEIQEIGSTKTQNVTINLGPFAQTGYAPVNWGPGETNDSTTVEPVLDGPYQPTTFNPPVDY
WMLLAPTAAGVVVEGTNNTDRWLATILVEPNVTSETRSYTLFGTQEQITIANASQTQWKFIDVVKTTQNGSYSQYGPLQS
TPKLYAVMKHNGKIYTYNGETPNVTTKYYSTTNYDSVNMTAFCDFYIIPREEESTCTEYINNGLPPIQNTRNIVPLALSA
RNIISHRAQANEDIVVSKTSLWKEMQYNRDITIRFKFASSIVKSGGLGYKWSEISFKPANYQYTYTRDGEEVTAHTTCSV
NGMNDFNFNGGSLPTDFVISRYEVIKENSYVYVDYWDDSQAFRNMVYVRSLAANLNSVICTGGDYSFALPVGQWPVMTGG
AVSLHSAGVTLSTQFTDFVSLNSLRFRFRLTVEEPSFSITRTRVSRLYGLPAANPNNGKEYYEVAGRFSLISLVPSNDDY
QTPITNSVTVRQDLERQLGELREEFNALSQEIAMSQLIDLALLPLDMFSMFSGIKSTIDAAKSMATSVMKKFKKSGLANS
VSTLTDSLSDAASSISRGASIRSVGSSASAWTDVSTQITDVSSSVSSISTQTSTISRRLRLKEMATQTEGMNFDDISAAV
LKTKIDRSTQISPNTLPDIVTEASEKFIPNRAYRVINNDEVFEAGTDGRFFAYRVETFDEIPFDVQKFADLVTDSPVISA
IIDFKTLKNLNDNYGISRQQAFNLLRSDPRVLREFINQDNPIIRNRIEQLIMQCRLIntermediate capsid protein VP6RV-ARVA/Monkey/United States/RRV/1975/G3P5B[3]0.04493476618LEVOMDVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIITMNGNEFQTGGIGNLPIRNWNFDFGLLGTTLLNLDANYVET
ARNTIDYFVDFVDNVCMDEMVRESQRNGIAPQSDSLRKLSGIKFKRINFDNSSEYIENWNLQNRRQRTGFTFHKPNIFPY
SASFTLNRSQPAHDNLMGTMWLNAGSEIQVAGFDYSCAINAPANIQQFEHIVQLRRVLTTATITLLPDAERFSFPRVINS
ADGATTWYFNPVILRPNNVEVEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLMRPPNMTPAVAALFPNAQPFEHHATV
GLTLRIESAVCESVLADASKTMLANVTSVRQEYAIPVGPVFPPGMNWTDLITNYSPSREDNLQRVFTVASIRSMLVKOuter capsid glycoprotein VP7Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])RVA/Monkey/United States/RRV/1975/G3P5B[3]0.03713653118Spodoptera frugiperdaLEVOMYGIEYTTVLTFLISLILLNYILKSLTRMMDFIIYRFLFIVVILSPLLKAQNYGINLPITGSMDTAYANSTQEETFLTST
LCLYYPTEAATEINDNSWKDTLSQLFLTKGWPTGSVYFKEYTDIASFSVDPQLYCDYNVVLMKYDATLQLDMSELADLIL
NEWLCNPMDITLYYYQQTDEANKWISMGSSCTIKVCPLNTQTLGIGCLTTDTATFEEVATAEKLVITDVVDGVNHKLDVT
TATCTIRNCKKLGPRENVAVIQVGGSDVLDITADPTTAPQTERMMRINWKKWWQVFYTVVDYVNQIIQAMSKRSRSLNSA
AFYYRI2-acetamido-2-deoxy-beta-D-glucopyranose0.00022120818NAGCALCIUM ION4.0078e-0554CAsingleParticleparticle1.28.020.02-Amino-2-(hydroxymethyl)propan-1,3-diolTris100.0NaClSodium Chloride1.0CaCl2Calcium ChlorideC-flat-1.2/1.3 4CCOPPER400CARBONHOLEYGLOW DISCHARGEAIRETHANE90298.15GATAN CRYOPLUNGE 34 ul sample volume, 4 sec blotting time. FEI POLARA 300FLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN3002.01.01.03.03.027500.040605.0NITROGENGATAN K2 SUMMIT (4k x 4k)COUNTING383837011-501410710.033.0114579whole viral particlesGctf4V7Q1C1FOURIER SPACE3.4FSC 0.143 CUT-OFFcisTEMReconstruction final map after classification of VP4 sub-particles. VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)167147PROJECTION MATCHINGcisTEMEuler angle determination of viral particles by projection matching to cryo-EM map from pdb 4V7QPROJECTION MATCHINGcisTEM3250000.0cisTEMClassification of VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)4V7QOTHERphenix.real_space_refineREALemd_21955_msk_1.mapemd_21955_half_map_2.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
320
320320
0
00320320320393.91998393.91998393.9199890.090.090.0XYZ-0.03385910.0600554721.0659577e-060.005757551.2311.2311.231half map 2emd_21955_half_map_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)