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- EMDB-20060: In situ structure of Rotavirus RNA-dependent RNA polymerase at tr... -

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Basic information

Entry
Database: EMDB / ID: EMD-20060
TitleIn situ structure of Rotavirus RNA-dependent RNA polymerase at transcript-elongated state
Map data
SampleRotavirus A
  • virus
  • (nucleic-acidNucleic acid) x 2
  • RNA-dependent RNA polymerase of rotavirus A
  • Inner capsid protein VP2
  • (ligand) x 2
Function / homology
Function and homology information


T=2 icosahedral viral capsid / viral inner capsid / viral genome replication / virion / RNA-directed RNA polymerase / viral nucleocapsid / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / nucleotide binding / RNA binding
RNA-directed RNA polymerase, luteovirus / Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Rotavirus VP1 C-terminal domain / Rotavirus VP2 protein / Viral RNA-directed RNA-polymerase / Viral RNA-directed RNA polymerase, 4-helical domain / RNA-directed RNA polymerase, reovirus / Rotavirus VP2
RNA-directed RNA polymerase / Inner capsid protein VP2
Biological speciesRotavirus A
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDing K / Chang T / Shen W / Roy P / Zhou ZH
CitationJournal: Nat Commun / Year: 2019
Title: In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release.
Authors: Ke Ding / Cristina C Celma / Xing Zhang / Thomas Chang / Wesley Shen / Ivo Atanasov / Polly Roy / Z Hong Zhou /
Abstract: Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron ...Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.
Validation ReportPDB-ID: 6ogz

SummaryFull reportAbout validation report
History
DepositionApr 3, 2019-
Header (metadata) releaseApr 10, 2019-
Map releaseMay 22, 2019-
UpdateMay 29, 2019-
Current statusMay 29, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6ogz
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6ogz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20060.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 264 pix.
= 353.76 Å
1.34 Å/pix.
x 264 pix.
= 353.76 Å
1.34 Å/pix.
x 264 pix.
= 353.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.09637474 - 0.13541356
Average (Standard dev.)0.0010679516 (±0.00976145)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 353.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z353.760353.760353.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.0960.1350.001

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Supplemental data

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Sample components

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Entire Rotavirus A

EntireName: Rotavirus A / Number of components: 7

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Component #1: virus, Rotavirus A

VirusName: Rotavirus A / Class: VIRION / Empty: No / Enveloped: No / Isolate: SPECIES
SpeciesSpecies: Rotavirus A

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Component #2: nucleic-acid, RNA (5'-R(P*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*...

nucleic acidName: RNA (5'-R(P*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UAUAUAUAUA UAUAUAUA
MassTheoretical: 5.673388 kDa
SourceSpecies: Rotavirus A

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Component #3: nucleic-acid, RNA (5'-R(P*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*...

nucleic acidName: RNA (5'-R(P*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*AP*UP*A)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AUAUAUAUAU AUAUAUA
MassTheoretical: 5.367222 kDa
SourceSpecies: Rotavirus A

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Component #4: protein, RNA-dependent RNA polymerase of rotavirus A

ProteinName: RNA-dependent RNA polymerase of rotavirus A / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 125.276305 kDa
SourceSpecies: Rotavirus A

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Component #5: protein, Inner capsid protein VP2

ProteinName: Inner capsid protein VP2 / Number of Copies: 10 / Recombinant expression: No
MassTheoretical: 103.425992 kDa
SourceSpecies: Rotavirus A

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Component #6: ligand, URIDINE 5'-TRIPHOSPHATE

LigandName: URIDINE 5'-TRIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.484141 kDa

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Component #7: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
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: 18 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 411438
3D reconstructionSoftware: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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