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- EMDB-31183: In situ structure of transcriptional enzyme complex and capsid sh... -

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Basic information

Entry
Database: EMDB / ID: EMD-31183
TitleIn situ structure of transcriptional enzyme complex and capsid shell protein of mammalian reovirus at initiation state
Map data
Sample
  • Virus: Mammalian orthoreovirus 3 Dearing
    • Protein or peptide: Minor core protein mu2
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • RNA: transcript RNA
    • RNA: RNA (60-MER)
  • Protein or peptide: Lambda 1
  • Protein or peptide: Lambda 1
  • Ligand: PHOSPHATE IONPhosphate
Function / homology
Function and homology information


viral inner capsid / host cytoskeleton / 7-methylguanosine mRNA capping / viral genome replication / viral capsid / viral nucleocapsid / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / structural molecule activity / RNA binding
Similarity search - Function
Reovirus minor core protein, Mu-2 / Reovirus minor core protein Mu-2 / Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus RNA-dependent RNA polymerase lambda 3 / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase / Lambda 1 / Minor core protein mu2
Similarity search - Component
Biological speciesMammalian orthoreovirus 3 / Mammalian orthoreovirus 3 Dearing
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhou ZH / Pan M
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1S10RR23057 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1S10OD018111 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: Nat Commun / Year: 2021
Title: Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins.
Authors: Muchen Pan / Ana L Alvarez-Cabrera / Joon S Kang / Lihua Wang / Chunhai Fan / Z Hong Zhou /
Abstract: Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious ...Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.
History
DepositionApr 11, 2021-
Header (metadata) releaseOct 20, 2021-
Map releaseOct 20, 2021-
UpdateOct 20, 2021-
Current statusOct 20, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7elh
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7elh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31183.png

Downloads & links

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Map

FileDownload / File: emd_31183.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.01811592 - 0.03685395
Average (Standard dev.)0.001506305 (±0.0039825174)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 428.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z428.000428.000428.000
α/β/γ90.00090.00090.000
start NX/NY/NZ336210602
NX/NY/NZ227193139
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0180.0370.002

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Supplemental data

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Sample components

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Entire : Mammalian orthoreovirus 3 Dearing

EntireName: Mammalian orthoreovirus 3 Dearing
Components
  • Virus: Mammalian orthoreovirus 3 Dearing
    • Protein or peptide: Minor core protein mu2
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • RNA: transcript RNA
    • RNA: RNA (60-MER)
  • Protein or peptide: Lambda 1
  • Protein or peptide: Lambda 1
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: Mammalian orthoreovirus 3 Dearing

SupramoleculeName: Mammalian orthoreovirus 3 Dearing / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #6 / NCBI-ID: 10886 / Sci species name: Mammalian orthoreovirus 3 Dearing / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Minor core protein mu2

MacromoleculeName: Minor core protein mu2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mammalian orthoreovirus 3
Molecular weightTheoretical: 83.331289 KDa
SequenceString: MAYIAVPAVV DSRSSEAIGL LESFGVDAGA DANDVSYQDH DYVLDQLQYM LDGYEAGDVI DALVHKNWLH HSVYCLLPPK SQLLEYWKS NPSAIPDNVD RRLRKRLMLK KDLRKDDEYN QLARAFKISD VYAPLISSTT SPMTMIQNLN QGEIVYTTTD R VIGARILL ...String:
MAYIAVPAVV DSRSSEAIGL LESFGVDAGA DANDVSYQDH DYVLDQLQYM LDGYEAGDVI DALVHKNWLH HSVYCLLPPK SQLLEYWKS NPSAIPDNVD RRLRKRLMLK KDLRKDDEYN QLARAFKISD VYAPLISSTT SPMTMIQNLN QGEIVYTTTD R VIGARILL YAPRKYYAST LSFTMTKCII PFGKEVGRVP HSRFNVGTFP SIATPKCFVM SGVDIESIPN EFIKLFYQRV KS VHANILN DISPQIVSDM INRKRLRVHT PSDRRAAQLM HLPYHVKRGA SHVDVYKVDV VDMLFEVVDV ADGLRNVSRK LTM HTVPVC ILEMLGIEIA DYCIRQEDGM LTDWFLLLTM LSDGLTDRRT HCQYLINPSS VPPDVILNIS ITGFINRHTI DVMP DIYDF VKPIGAVLPK GSFKSTIMRV LDSISILGIQ IMPRAHVVDS DEVGEQMEPT FEQAVMEIYK GIAGVDSLDD LIKWV LNSD LIPHDDRLGQ LFQAFLPLAK DLLAPMARKF YDNSMSEGRL LTFAHADSEL LNANYFGHLL RLKIPYITEV NLMIRK NRE GGELFQLVLS YLYKMYATSA QPKWFGSLLR LLICPWLHME KLIGEADPAS TSAEIGWHIP REQLMQDGWC GCEDGFI PY VSIRAPRLVI EELMEKNWGQ YHAQVIVTDQ LVVGEPRRVS AKAVIKGNHL PVKLVSRFAC FTLTAKYEMR LSCGHSTG R GAAYSARLAF RSDLA

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Macromolecule #2: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Mammalian orthoreovirus 3
Molecular weightTheoretical: 142.449016 KDa
SequenceString: MSSMILTQFG PFIESISGIT DQSNDVFEDA AKAFSMFTRS DVYKALDEIP FSDDAMLPIP PTIYTKPSHD SYYYIDALNR VRRKTYQGP DDVYVPNCSI VELLEPHETL TSYGRLSEAI ENRAKDGDSQ ARIATTYGRI AESQARQIKA PLEKFVLALL V AEAGGSLY ...String:
MSSMILTQFG PFIESISGIT DQSNDVFEDA AKAFSMFTRS DVYKALDEIP FSDDAMLPIP PTIYTKPSHD SYYYIDALNR VRRKTYQGP DDVYVPNCSI VELLEPHETL TSYGRLSEAI ENRAKDGDSQ ARIATTYGRI AESQARQIKA PLEKFVLALL V AEAGGSLY DPVLQKYDEI PDLSHNCPLW CFREICRHIS GPLPDRAPYL YLSAGVFWLM SPRMTSAIPP LLSDLVNLAI LQ QTAGLDP SLVKLGVQIC LHAAASSSYA WFILKTKSIF PQNTLHSMYE SLEGGYCPNL EWLEPRSDYK FMYMGVMPLS AKY ARSAPS NDKKARELGE KYGLSSVVGE LRKRTKTYVK HDFASVRYIR DAMACTSGIF LVRTPTETVL QEYTQSPEIK VPIP QKDWT GPIGEIRILK DTTSSIARYL YRTWYLAAAR MAAQPRTWDP LFQAIMRSQY VTARGGSGAA LRESLYAINV SLPDF KGLP VKAATKIFQA AQLANLPFSH TSVAILADTS MGLRNQVQRR PRSIMPLNVP QQQVSAPHTL TADYINYHMN LSTTSG SAV IEKVIPLGVY ASSPPNQSIN IDISACDASI TWDFFLSVIM AAIHEGVASS SIGKPFMGVP ASIVNDESVV GVRAARP IS GMQNMIQHLS KLYKRGFSYR VNDSFSPGND FTHMTTTFPS GSTATSTEHT ANNSTMMETF LTVWGPEHTD DPDVLRLM K SLTIQRNYVC QGDDGLMIID GTTAGKVNSE TIQKMLELIS KYGEEFGWKY DIAYDGTAEY LKLYFIFGCR IPNLSRHPI VGKERANSSA EEPWPAILDQ IMGVFFNGVH DGLQWQRWIR YSWALCCAFS RQRTMIGESV GYLQYPMWSF VYWGLPLVKA FGSDPWIFS WYMPTGDLGM YSWISLIRPL MTRWMVANGY VTDRCSPVFG NADYRRCFNE LKLYQGYYMA QLPRNPKKSG R AAPREVRE QFTQALSDYL MQNPELKSRV LRGRSEWEKY GAGIIHNPPS LFDVPHKWYQ GAQEAAIATR EELAEMDETL MR ARRHSYS SFSKLLEAYL LVKWRMCEAR EPSVDLRLPL CAGIDPLNSD PFLKMVSVGP MLQSTRKYFA QTLFMAKTVS GLD VNAIDS ALLRLRTLGA DKKALTAQLL MVGLQESEAD ALAGKIMLQD VNTVQLARVV NLAVPDTWMS LDFDSMFKHH VKLL PKDGR HLNTDIPPRM GWLRAILRFL GAGMVMTATG VAVDIYLEDI HGGGRSLGQR FMTWMRQEGR SA

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Macromolecule #4: Lambda 1

MacromoleculeName: Lambda 1 / type: protein_or_peptide / ID: 4 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Mammalian orthoreovirus 3
Molecular weightTheoretical: 122.842859 KDa
SequenceString: YQCHVCSAVL FSPLDLDAHV ASHGLHGNMT LTSSDIQRHI TEFISSWQNH PIVQVSADVE NKKTAQLLHA DTPRLVTWDA GLCTSFKIV PIVPAQVPQD VLAYTFFTSS YAIQSPFPEA AVSRIVVHTR WASNVDFDRD SSVIMAPPTE NNIHLFKQLL N TETLSVRG ...String:
YQCHVCSAVL FSPLDLDAHV ASHGLHGNMT LTSSDIQRHI TEFISSWQNH PIVQVSADVE NKKTAQLLHA DTPRLVTWDA GLCTSFKIV PIVPAQVPQD VLAYTFFTSS YAIQSPFPEA AVSRIVVHTR WASNVDFDRD SSVIMAPPTE NNIHLFKQLL N TETLSVRG ANPLMFRANV LHMLLEFVLD NLYLNRHTGF SQDHTPFTEG ANLRSLPGPD AEKWYSIMYP TRMGTPNVSK IC NFVASCV RNRVGRFDRA QMMNGAMSEW VDVFETSDAL TVSIRGRWMA RLARMNINPT EIEWALTECA QGYVTVTSPY APS VNRLMP YRISNAERQI SQIIRIMNIG NNATVIQPVL QDISVLLQRI SPLQIDPTII SNTMSTVSES TTQTLSPASS ILGK LRPSN SDFSSFRVAL AGWLYNGVVT TVIDDSSYPK DGGSVTSLEN LWDFFILALA LPLTTDPCAP VKAFMTLANM MVGFE TIPM DNQIYTQSRR ASAFSTPHTW PRCFMNIQLI SPIDAPILRQ WAEIIHRYWP NPSQIRYGAP NVFGSANLFT PPEVLL LPI DHQPANVTTP TLDFTNELTN WRARVCELMK NLVDNQRYQP GWTQSLVSSM RGTLDKLKLI KSMTPMYLQQ LAPVELA VI APMLPFPPFQ VPYVRLDRDR VPTMVGVTRQ SRDTITQPAL SLSTTNTTVG VPLALDARAI TVALLSGKYP PDLVTNVW Y ADAIYPMYAD TEVFSNLQRD MITCEAVQTL VTLVAQISET QYPVDRYLDW IPSLRASAAT AATFAEWVNT SMKTAFDLS DMLLEPLLSG DPRMTQLAIQ YQQYNGRTFN IIPEMPGSVI ADCVQLTAEV FNHEYNLFGI ARGDIIIGRV QSTHLWSPLA PPPDLVFDR DTPGVHIFGR DCRISFGMNG AAPMIRDETG LMVPFEGNWI FPLALWQMNT RYFNQQFDAW IKTGELRIRI E MGAYPYML HYYDPRQYAN AWNLTSAWLE EITPTSIPSV PFMVPISSDH DISSAPAVQY IISTEYNDRS LFCTNSSSPQ TI AGPDKHI PVERYNILTN PDAPPTQIQL PEVVDLYNVV TRYAYETPPI TAVVMGVP

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Macromolecule #5: Lambda 1

MacromoleculeName: Lambda 1 / type: protein_or_peptide / ID: 5 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mammalian orthoreovirus 3
Molecular weightTheoretical: 19.112762 KDa
SequenceString:
MKRIPRKTKG KSSGKGNDST ERADDGSSQL RDKQNNKAGP ATTEPGTSNR EQYKARPGIA SVQRATESAE MPMKNNDEGT PDKKGNTKG DLVNEHSEAK DEADEATKKQ AKDTDKSKAQ VTYSDTGINN ANELSRSGNV DNEGGSNQKP MSTRIAEATS A IVSKHPAR VGLPPTASSG HG

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Macromolecule #3: transcript RNA

MacromoleculeName: transcript RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Mammalian orthoreovirus 3 Dearing
Molecular weightTheoretical: 1.263842 KDa
SequenceString:
AAGC

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Macromolecule #6: RNA (60-MER)

MacromoleculeName: RNA (60-MER) / type: rna / ID: 6 / Number of copies: 8
Source (natural)Organism: Mammalian orthoreovirus 3 Dearing
Molecular weightTheoretical: 19.016188 KDa
SequenceString:
AUAUAUAUAU AUAUAUAUAU AUAUAUAUAU AUAUAUAUAU AUAUAUAUAU AUAUAUAUAU

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Macromolecule #7: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Sphere
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 102966
FSC plot (resolution estimation)

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