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- PDB-6po2: In situ structure of BTV RNA-dependent RNA polymerase in BTV core -

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Basic information

Entry
Database: PDB / ID: 6po2
TitleIn situ structure of BTV RNA-dependent RNA polymerase in BTV core
Components
  • Inner core structural protein VP3
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / TRANSFERASE / RNA dependent RNA polymerase
Function / homology
Function and homology information


viral genome replication / RNA-directed RNA polymerase / nucleotide binding / RNA-dependent RNA polymerase activity / DNA-templated transcription / structural molecule activity / RNA binding
Similarity search - Function
RNA-dependent RNA polymerase, orbiviral / Orbivirus RNA-dependent RNA polymerase (VP1) / Inner layer core protein VP3, Orbivirus / Orbivirus VP3 (T2) protein / Inner layer core protein VP3, Reovirus / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Core protein VP3 / RNA-directed RNA polymerase
Similarity search - Component
Biological speciesBluetongue virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHe, Y. / Shivakoti, S. / Ding, K. / Cui, Y. / Roy, P. / Zhou, Z.H.
Funding support United States, United Kingdom, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI045000 United States
Wellcome Trust100218 United Kingdom
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating.
Authors: Yao He / Sakar Shivakoti / Ke Ding / Yanxiang Cui / Polly Roy / Z Hong Zhou /
Abstract: Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the , a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA- ...Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the , a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.
History
DepositionJul 3, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 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-directed RNA polymerase
B: Inner core structural protein VP3
C: Inner core structural protein VP3
D: Inner core structural protein VP3
E: Inner core structural protein VP3
F: Inner core structural protein VP3
G: Inner core structural protein VP3
H: Inner core structural protein VP3
I: Inner core structural protein VP3
J: Inner core structural protein VP3
K: Inner core structural protein VP3


Theoretical massNumber of molelcules
Total (without water)1,184,03111
Polymers1,184,03111
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area80590 Å2
ΔGint-347 kcal/mol
Surface area398650 Å2

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Components

#1: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase


Mass: 149926.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bluetongue virus 1 / References: UniProt: W0G557, RNA-directed RNA polymerase
#2: Protein
Inner core structural protein VP3 / Inner capsid protein VP3


Mass: 103410.508 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bluetongue virus 1 / Gene: VP3 / Production host: Bluetongue virus 1 / References: UniProt: Q1AE73

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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: Bluetongue virus 1Bluetongue disease / Type: VIRUS / Entity ID: all / Source: NATURAL
Source (natural)Organism: Bluetongue virus 1
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Buffer solutionpH: 8.8
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: 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 / Cs: 2.7 mm
Image recordingElectron dose: 32 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150346 / Symmetry type: POINT

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