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- PDB-6m99: In situ structure of transcriptional enzyme complex and asymmetri... -

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Basic information

Entry
Database: PDB / ID: 6m99
TitleIn situ structure of transcriptional enzyme complex and asymmetric inner capsid protein of aquareovirus at primed state
Components
  • Putative core protein NTPase/VP5
  • VP2
  • VP3
KeywordsVIRAL PROTEIN / Reovirus / transcriptional enzyme complex / polymerase / RdRp / NTPase
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 helicase activity / hydrolase activity / RNA helicase ...viral inner capsid / host cytoskeleton / 7-methylguanosine mRNA capping / viral genome replication / viral capsid / viral nucleocapsid / host cell cytoplasm / RNA helicase activity / hydrolase activity / RNA helicase / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / structural molecule activity / RNA binding / metal ion 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 / Inner capsid protein lambda-1/ VP3 / C2H2-type zinc finger / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / zinc finger / Zinc finger C2H2 type domain profile. ...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 / Inner capsid protein lambda-1/ VP3 / C2H2-type zinc finger / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / zinc finger / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
PHOSPHATE ION / Putative core protein NTPase/VP5 / RNA helicase / RNA-directed RNA polymerase
Similarity search - Component
Biological speciesGrass carp reovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDing, K. / Zhou, Z.H.
Funding support United States, 8items
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 General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DE025567 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)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: J Virol / Year: 2018
Title: Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.
Authors: Ke Ding / Lisa Nguyen / Z Hong Zhou /
Abstract: Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The structures of the transcriptional ...Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions. Viruses in the family are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.
History
DepositionAug 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 5, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Assembly

Deposited unit
A: VP2
B: Putative core protein NTPase/VP5
C: VP3
D: VP3
E: VP3
F: VP3
G: VP3
H: VP3
I: VP3
J: VP3
K: VP3
L: VP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,544,26014
Polymers1,544,10012
Non-polymers1602
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area98500 Å2
ΔGint-455 kcal/mol
Surface area451960 Å2

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Components

#1: Protein VP2 / RNA dependent RNA polymerase


Mass: 141685.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Grass carp reovirus / References: UniProt: Q9E3V9
#2: Protein Putative core protein NTPase/VP5


Mass: 80381.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Grass carp reovirus / References: UniProt: Q8JU68
#3: Protein
VP3 / Inner capsid protein


Mass: 132203.312 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Grass carp reovirus / References: UniProt: Q9E3V8
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has protein modificationY

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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: transcriptional enzyme complex and asymmetric inner capsid protein
Type: VIRUS / Entity ID: #1-#3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Grass carp reovirus
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Ctenopharyngodon idella
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELD
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73472 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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