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- EMDB-20086: In situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP) -

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Basic information

Entry
Database: EMDB / ID: EMD-20086
TitleIn situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP)
Map data
SampleRhesus rotavirus
  • virus
  • Inner capsid protein VP2
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
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
Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / Rotavirus VP2 / RNA-directed RNA polymerase, reovirus / RNA-directed RNA polymerase, luteovirus
RNA-directed RNA polymerase / Inner capsid protein VP2
Biological speciesRotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3]) / RV-A (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsJenni S / Salgado EN / Herrmann T / Li Z / Grant T / Grigorieff N / Trapani S / Estrozi LF / Harrison SC
Funding support United States, 2 items
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.
Validation ReportPDB-ID: 6oj3

SummaryFull reportAbout validation report
History
DepositionApr 10, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 2019-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oj3
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20086.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 300 pix.
= 369. Å
1.23 Å/pix.
x 300 pix.
= 369. Å
1.23 Å/pix.
x 300 pix.
= 369. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 2 / Movie #1: 2
Minimum - Maximum-6.1107807 - 9.9597025
Average (Standard dev.)0.018543862 (±0.42538026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 369.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z369.000369.000369.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-420-29
NX/NY/NZ887886
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-6.1119.9600.019

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Supplemental data

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Segmentation: #1

Fileemd_20086_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: filtered, B-sharpened

Fileemd_20086_additional_1.map
Annotationfiltered, B-sharpened
Projections & Slices
AxesZYX

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Additional map: filtered

Fileemd_20086_additional_2.map
Annotationfiltered
Projections & Slices
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Half map: half-map 1, unmodified

Fileemd_20086_half_map_1.map
Annotationhalf-map 1, unmodified
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half-map 2, unmodified

Fileemd_20086_half_map_2.map
Annotationhalf-map 2, unmodified
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Rhesus rotavirus

EntireName: Rhesus rotavirus / Details: TLP / Number of components: 3

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Component #1: virus, Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])

VirusName: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Class: VIRION / Details: TLP / Empty: No / Enveloped: No / Isolate: SEROTYPE
SpeciesSpecies: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]

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Component #2: protein, Inner capsid protein VP2

ProteinName: Inner capsid protein VP2 / Number of Copies: 10 / Recombinant expression: No
MassTheoretical: 103.425992 kDa
SourceSpecies: RV-A (virus) / Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]

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Component #3: protein, RNA-directed RNA polymerase

ProteinName: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 125.276305 kDa
SourceSpecies: RV-A (virus) / Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 64 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 / Applied symmetry: C1 (asymmetric) / Number of projections: 30606
3D reconstructionSoftware: cisTEM / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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