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- PDB-6oj3: In situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP) -

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Basic information

Entry
Database: PDB / ID: 6oj3
TitleIn situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP)
Components
  • Inner capsid protein VP2
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN/TRANSFERASE / Rotavirus / RNA-dependent RNA polymerase / VP1 / VP2 / VIRAL PROTEIN-TRANSFERASE complex
Function / homology
Function and homology information


T=2 icosahedral viral capsid / viral inner capsid / viral genome replication / viral nucleocapsid / RNA-directed RNA polymerase / RNA-directed 5'-3' RNA polymerase activity / DNA-templated transcription / nucleotide binding / RNA binding
Similarity search - Function
Rotavirus VP2 protein / Rotavirus VP2 / Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP1 C-terminal domain / Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / RNA-directed RNA polymerase, luteovirus / Viral RNA-directed RNA-polymerase / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase / Inner capsid protein VP2
Similarity search - Component
Biological speciesRotavirus A
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsJenni, S. / Salgado, E.N. / Herrmann, T. / Li, Z. / Grant, T. / Grigorieff, N. / Trapani, S. / Estrozi, L.F. / Harrison, S.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)CA-13202 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Mol Biol / Year: 2019
Title: In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.
Authors: Simon Jenni / Eric N Salgado / Tobias Herrmann / Zongli Li / Timothy Grant / Nikolaus Grigorieff / Stefano Trapani / Leandro F Estrozi / Stephen C Harrison /
Abstract: Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of ...Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg, and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome.
History
DepositionApr 10, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Aug 28, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Nov 27, 2019Group: Data collection / Category: em_software / Item: _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Inner capsid protein VP2
B: Inner capsid protein VP2
C: Inner capsid protein VP2
D: Inner capsid protein VP2
E: Inner capsid protein VP2
F: Inner capsid protein VP2
G: Inner capsid protein VP2
H: Inner capsid protein VP2
I: Inner capsid protein VP2
J: Inner capsid protein VP2
P: RNA-directed RNA polymerase


Theoretical massNumber of molelcules
Total (without water)1,159,53611
Polymers1,159,53611
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area70800 Å2
ΔGint-135 kcal/mol
Surface area355210 Å2

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Components

#1: Protein
Inner capsid protein VP2 / VP2A


Mass: 103425.992 Da / Num. of mol.: 10 / Source method: isolated from a natural source
Source: (natural) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Cell line: MA104 / Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3] / References: UniProt: B3F2X3
#2: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase / Protein VP1


Mass: 125276.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Cell line: MA104 / Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3] / References: UniProt: B3F2X2, RNA-directed RNA polymerase

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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: Rhesus rotavirus / Type: VIRUS / Details: TLP / Entity ID: all / Source: NATURAL
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Details of virusEmpty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: cisTEM / Version: 1.01 / Category: 3D reconstruction / Details: FrealignX
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30606 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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