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- EMDB-20159: Rotavirus A-VP3-8mer ssRNA complex (RVA-VP3-RNA) -

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Basic information

Entry
Database: EMDB / ID: EMD-20159
TitleRotavirus A-VP3-8mer ssRNA complex (RVA-VP3-RNA)
Map dataRotavirus-A VP3 tetramer binding with 8-mer ssRNA
Sample
  • Complex: VP3-8-mer ssRNA complex
Biological speciesRotavirus A
Methodsingle particle reconstruction / cryo EM / Resolution: 11.8 Å
AuthorsKumar D / Yu X
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI36040 United States
Robert A. Welch FoundationQ1279 United States
Welch FoundationQ-1967-20180324 United States
CitationJournal: Sci Adv / Year: 2020
Title: 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity.
Authors: Dilip Kumar / Xinzhe Yu / Sue E Crawford / Rodolfo Moreno / Joanita Jakana / Banumathi Sankaran / Ramakrishnan Anish / Soni Kaundal / Liya Hu / Mary K Estes / Zhao Wang / B V Venkataram Prasad /
Abstract: In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 ...In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo-electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.
History
DepositionApr 25, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseApr 29, 2020-
UpdateApr 29, 2020-
Current statusApr 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20159.png

Downloads & links

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Map

FileDownload / File: emd_20159.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRotavirus-A VP3 tetramer binding with 8-mer ssRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.74 Å/pix.
x 150 pix.
= 261. Å		1.74 Å/pix.
x 150 pix.
= 261. Å		1.74 Å/pix.
x 150 pix.
= 261. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.0068679973 - 0.03403599
Average (Standard dev.)0.00036393746 (±0.0030664506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 261.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.741.741.74
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z261.000261.000261.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0070.0340.000

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Supplemental data

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Sample components

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Entire : VP3-8-mer ssRNA complex

EntireName: VP3-8-mer ssRNA complex
Components
  • Complex: VP3-8-mer ssRNA complex

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Supramolecule #1: VP3-8-mer ssRNA complex

SupramoleculeName: VP3-8-mer ssRNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: VP3 tetramer binding with 8-mer ssRNA
Source (natural)Organism: Rotavirus A
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7
GridSupport film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 3200FSC
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 40000

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Image processing

Particle selectionNumber selected: 210818
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1.0) / Number images used: 23715
Initial angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 2.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.1.0)

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