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- EMDB-9050: In situ structure of transcriptional enzyme complex and asymmetri... -

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Entry
Database: EMDB / ID: 9050
TitleIn situ structure of transcriptional enzyme complex and asymmetric inner capsid protein of aquareovirus at primed state
Map dataGrass Carp reovirus TEC structure at primed state
Sampletranscriptional enzyme complex and asymmetric inner capsid protein:
virus / VP2 / Putative core protein NTPase/VP5 / VP3 / (ligand) x 2
Function / homologyReovirus RNA-dependent RNA polymerase lambda 3 / Reovirus minor core protein Mu-2 / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / RNA-directed RNA polymerase, reovirus / Reovirus minor core protein, Mu-2 / Reovirus RNA-dependent RNA polymerase lambda 3 / Zinc finger C2H2-type / viral genome replication ...Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus minor core protein Mu-2 / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / RNA-directed RNA polymerase, reovirus / Reovirus minor core protein, Mu-2 / Reovirus RNA-dependent RNA polymerase lambda 3 / Zinc finger C2H2-type / viral genome replication / viral capsid / nucleic acid binding / RNA-directed 5'-3' RNA polymerase activity / viral nucleocapsid / structural molecule activity / RNA binding / Putative core protein NTPase/VP5 / VP3 / VP2
Function and homology information
SourceGrass carp reovirus
Methodsingle particle reconstruction / cryo EM / 3.4 Å resolution
AuthorsDing K / Zhou ZH
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.
Validation ReportPDB-ID: 6m99

SummaryFull reportAbout validation report
DateDeposition: Aug 23, 2018 / Header (metadata) release: Sep 5, 2018 / Map release: Sep 5, 2018 / Last update: Sep 5, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6m99
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6m99
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9050.map.gz (map file in CCP4 format, 108001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.33 Å/pix.
= 399. Å
300 pix
1.33 Å/pix.
= 399. Å
300 pix
1.33 Å/pix.
= 399. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density
Contour Level:0.0194 (by author), 0.0194 (movie #1):
Minimum - Maximum-0.050881516 - 0.102366626
Average (Standard dev.)0.0029607322 (0.013269705)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin0.0.0.
Limit299.299.299.
Spacing300300300
CellA=B=C: 399.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z399.000399.000399.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0510.1020.003

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Supplemental data

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Sample components

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Entire transcriptional enzyme complex and asymmetric inner capsid protein

EntireName: transcriptional enzyme complex and asymmetric inner capsid protein
Number of components: 6

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Component #1: virus, Grass carp reovirus

VirusName: Grass carp reovirus / Class: VIRION / Empty: No / Enveloped: No / Isolate: STRAIN
SpeciesSpecies: Grass carp reovirus
Source (natural)Host Species: Ctenopharyngodon idella (grass carp)

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Component #2: protein, VP2

ProteinName: VP2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 141.685438 kDa
SourceSpecies: Grass carp reovirus

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Component #3: protein, Putative core protein NTPase/VP5

ProteinName: Putative core protein NTPase/VP5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 80.381516 kDa
SourceSpecies: Grass carp reovirus

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Component #4: protein, VP3

ProteinName: VP3 / Number of Copies: 10 / Recombinant expression: No
MassTheoretical: 132.203312 kDa
SourceSpecies: Grass carp reovirus

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Component #5: ligand, PHOSPHATE ION

LigandName: PHOSPHATE IONPhosphate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.497105 MDa

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Component #6: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 73472
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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