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- PDB-6tz1: In situ structure of BmCPV RNA-dependent RNA polymerase at early-... -

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Basic information

Entry
Database: PDB / ID: 6tz1
TitleIn situ structure of BmCPV RNA-dependent RNA polymerase at early-elongation state
Components
  • Non-template RNA (5'-D(*(GTA))-R(P*GP*UP*A)-3')
  • RNA-dependent RNA Polymerase
  • Template RNA (5'-R(P*AP*GP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Transcript (5'-R(P*UP*UP*UP*UP*UP*CP*CP*CP*U)-3')
  • Viral structural protein 4
KeywordsVIRAL PROTEIN / TRANSFERASE/RNA / RdRp-RNA complex / Early-elongation / Cap-binding / TRANSFERASE-RNA complex
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 / RNA (> 10) / RNA dependent RNA Polymerase / Viral structural protein 4
Function and homology information
Biological speciesBombyx mori cytoplasmic polyhedrosis virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsCui, Y.X. / Zhang, Y.N. / Sun, J.C. / Zhou, Z.H.
Funding support China, United States, 8items
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 9, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Author supporting evidence / Database references / Category: citation / citation_author / pdbx_audit_support
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: RNA-dependent RNA Polymerase
B: Viral structural protein 4
M: Transcript (5'-R(P*UP*UP*UP*UP*UP*CP*CP*CP*U)-3')
N: Non-template RNA (5'-D(*(GTA))-R(P*GP*UP*A)-3')
T: Template RNA (5'-R(P*AP*GP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)210,6835
Polymers210,6835
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13800 Å2
ΔGint-56 kcal/mol
Surface area68480 Å2

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Components

#1: Protein RNA-dependent RNA Polymerase /


Mass: 139007.125 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Bombyx mori cytoplasmic polyhedrosis virus
References: UniProt: Q993A4, RNA-directed RNA polymerase
#2: Protein Viral structural protein 4


Mass: 63683.738 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Bombyx mori cytoplasmic polyhedrosis virus
References: UniProt: Q9IR43
#3: RNA chain Transcript (5'-R(P*UP*UP*UP*UP*UP*CP*CP*CP*U)-3')


Mass: 2710.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori cytoplasmic polyhedrosis virus
#4: RNA chain Non-template RNA (5'-D(*(GTA))-R(P*GP*UP*A)-3')


Mass: 1705.046 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori cytoplasmic polyhedrosis virus
#5: RNA chain Template RNA (5'-R(P*AP*GP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 3576.306 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bombyx mori cytoplasmic polyhedrosis virus
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bombyx mori cypovirus 1 / Type: VIRUS / Entity ID: all / Source: NATURAL
Source (natural)Organism: Bombyx mori cytoplasmic polyhedrosis virus
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Bombyx mori
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43807 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614575
ELECTRON MICROSCOPYf_angle_d0.86819852
ELECTRON MICROSCOPYf_dihedral_angle_d10.3858656
ELECTRON MICROSCOPYf_chiral_restr0.0532236
ELECTRON MICROSCOPYf_plane_restr0.0062442

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