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- EMDB-20581: In situ structure of BmCPV RNA dependent RNA polymerase at quiesc... -

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Basic information

Entry
Database: EMDB / ID: EMD-20581
TitleIn situ structure of BmCPV RNA dependent RNA polymerase at quiescent state
Map dataSub-particle reconstruction of BmCPV in the quiescent state
Sample
  • Virus: Bombyx mori cytoplasmic polyhedrosis virus
    • Protein or peptide: RNA-dependent RNA Polymerase
    • Protein or peptide: Viral structural protein 4
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE
KeywordsRdRp / VIRAL PROTEIN / TRANSFERASE
Function / homologyRNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / viral genome replication / RNA-dependent RNA polymerase activity / RNA binding / RNA dependent RNA Polymerase / Viral structural protein 4
Function and homology information
Biological speciesBombyx mori cytoplasmic polyhedrosis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsCui YX / Zhang YN
Funding support China, United States, 8 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31672489 China
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)1S10RR23057 United States
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116792 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Conservative transcription in three steps visualized in a double-stranded RNA virus.
Authors: Yanxiang Cui / Yinong Zhang / Kang Zhou / Jingchen Sun / Z Hong Zhou /
Abstract: Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV) ...Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8-3.5 Å) RdRp-RNA in situ structures-representing quiescent, initiation, early elongation, elongation and abortive states-under seven experimental conditions of CPV. We observed the 'Y'-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.
History
DepositionAug 8, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseNov 20, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ty8
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ty8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20581.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-particle reconstruction of BmCPV in the quiescent state
Voxel sizeX=Y=Z: 1.062 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.057999387 - 0.10192888
Average (Standard dev.)0.0019910082 (±0.008718651)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.872 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0621.0621.062
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.872271.872271.872
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0580.1020.002

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Supplemental data

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Sample components

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Entire : Bombyx mori cytoplasmic polyhedrosis virus

EntireName: Bombyx mori cytoplasmic polyhedrosis virus
Components
  • Virus: Bombyx mori cytoplasmic polyhedrosis virus
    • Protein or peptide: RNA-dependent RNA Polymerase
    • Protein or peptide: Viral structural protein 4
  • Ligand: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE

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Supramolecule #1: Bombyx mori cytoplasmic polyhedrosis virus

SupramoleculeName: Bombyx mori cytoplasmic polyhedrosis virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / NCBI-ID: 110829
Sci species name: Bombyx mori cytoplasmic polyhedrosis virus
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Bombyx mori (domestic silkworm)

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Macromolecule #1: RNA-dependent RNA Polymerase

MacromoleculeName: RNA-dependent RNA Polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bombyx mori cytoplasmic polyhedrosis virus
Molecular weightTheoretical: 139.007125 KDa
SequenceString: MLPNTELYNT IFSETRKFTR ESFKEIEHLT AKLANDRVAR HDFLFNNSIA LISDYSGEDS NGNQLQATVT IPNEITNPKE YDPSDYPLA EDESFFKQGH KYDYLVTFRA GSLTNTYEPK TKMYKLHAAL DKLMHVKQRK SRFADLWREL CAVIASLDVW Y QTTNYPLR ...String:
MLPNTELYNT IFSETRKFTR ESFKEIEHLT AKLANDRVAR HDFLFNNSIA LISDYSGEDS NGNQLQATVT IPNEITNPKE YDPSDYPLA EDESFFKQGH KYDYLVTFRA GSLTNTYEPK TKMYKLHAAL DKLMHVKQRK SRFADLWREL CAVIASLDVW Y QTTNYPLR TYVKLLFHKG DEFPFYESPS QDKIIFNDKS VASILPTFVY TCCQVGTAIM SGILTHVESI VAMNHFLHCA KD SYIDEKL KIKGIGRSWY QEALHNVGRA TVPVWSQFNE VIGHRTKTTS EPHFVSSTFI SLRAKRAELL YPEFNEYINR ALR LSKTQN DVANYYAACR AMTNDGTFLA TLTELSLDAA VFPRIEQRLV TRPAVLMSNT RHESLKQKYA NGVGSIAQSY LSSF TDEIA KRVNGIHHDE AWLNFLTTSS PGRKLTEIEK LEVGGDVAAW SNSRIVMQAV FAREYRTPER IFKSLKAPIK LVERQ QSDR RQRAISGLDN DRLFLSFMPY TIGKQIYDLN DNAAQGKQAG NAFDIGEMLY WTSQRNVLLS SIDVAGMDAS VTTNTK DIY NTFVLDVASK CTVPRFGPYY AKNMEVFEVG KRQSQVKYVN AAWQACALEA ANSQTSTSYE SEIFGQVKNA EGTYPSG RA DTSTHHTVLL QGLVRGNELK RASDGKNSCL TTIKILGDDI MEIFQGNEND THDHAVSNAS ILNESGFATT AELSQNSI V LLQQLVVNGT FWGFADRISL WTREDTKDIG RLNLAMMELN ALIDDLLFRV RRPEGLKMLG FFCGAICLRR FTLSVDNKL YDSTYNNLSK YMTLVKYDKN PDFDSTLMSL ILPLAWLFMP RGGEYPAYPF ERRDGTFTED ESMFTARGAY KRRLLYDVSN IREMIQQNS MVLDDDLLHE YGFTGALLLI DLNILDLIDE VKKEDISPVK VNELATSLEQ LGKLGEREKS RRAASDLKIR G HALSNDIV YGYGLQEKIQ KSAMATKETT VQSKRVSSRL HEVIVAKTRD YKIPTMPADA LHLYEFEVED VTVDLLPHAK HT SYSNLAY NMSFGSDGWF AFALLGGLDR SANLLRLDVA SIRGNYHKFS YDDPVFKQGY KIYKSDATLL NDFFVAISAG PKE QGILLR AFAYYSLYGN VEYHYVLSPR QLFFLSDNPV SAERLVRIPP SYYVSTQCRA LYNIFSYLHI LRSITSNQGK RLGM VLHPG LIAYVRGTSQ GAILPEADNV

UniProtKB: RNA dependent RNA Polymerase

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Macromolecule #2: Viral structural protein 4

MacromoleculeName: Viral structural protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bombyx mori cytoplasmic polyhedrosis virus
Molecular weightTheoretical: 63.683738 KDa
SequenceString: MFAIDPLKHS KLYEEYGLYL RPHQINQEIK PTTIKKKELA PTIRSIKYAS LIHSMLAKHA ARHNGTLINP RMYADMITLG NTKVTVTKG TPKAQIDTLK MNGLTVVSKS RRNNKKKPVS DTTATIDENT DDIVTYKALT EMSTLIESFR LPSGLALIIF D DEKYQSLI ...String:
MFAIDPLKHS KLYEEYGLYL RPHQINQEIK PTTIKKKELA PTIRSIKYAS LIHSMLAKHA ARHNGTLINP RMYADMITLG NTKVTVTKG TPKAQIDTLK MNGLTVVSKS RRNNKKKPVS DTTATIDENT DDIVTYKALT EMSTLIESFR LPSGLALIIF D DEKYQSLI PNYINQLIAY TQPHIIPTWQ GIADFSDTYL RSYFKRPFEL TASNLAAPQK YNLSPMTRSI FNNTGREDAV IR KLYGYGE YVFIRYEGCL ITWTGIYGEV TMMVNLSKRD LGLDVGDDYL KEYKKLLFYG VITDAIPSGI SARSTIMKIS PHK MMNPSG GALAVLSKFL EAVVSTNVIN ATLVVYAEKG AGKTSFLSTY AEQLSLASGQ VVGHLSSDAY GRWLAKNKDV EEPS FAYDY VLSLDTDDNE SYYEQKASEL LISHGISEVA QYELLSVRKK IKMMDEMNEV LIAQLENADT HSERNFYYMV STGKT TPRT LIVEGHFNAQ DATIARTDTT VLLRTINDTT QAMRDRQRGG VVQLFLRDTY YRLLPALHTT VYPFEMLESI RRWKWV H

UniProtKB: Viral structural protein 4

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Macromolecule #3: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE

MacromoleculeName: P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GTA
Molecular weightTheoretical: 787.441 Da
Chemical component information

ChemComp-GTA:
P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE

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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: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE-PROPANE

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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 QUANTUM (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 323500

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