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- EMDB-16769: In situ STA of rotavirus DLP (penton) -

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Basic information

Entry
Database: EMDB / ID: EMD-16769
TitleIn situ STA of rotavirus DLP (penton)
Map dataMain map
Sample
  • Virus: Rotavirus A
Biological speciesRotavirus A
Methodsubtomogram averaging / cryo EM / Resolution: 10.2 Å
AuthorsShah PNM / Stuart DI
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CAMS Innovation Fund for Medical Sciences (CIFMS)2018-I2M-2-002 United Kingdom
Wellcome Trust03141/Z/16/Z United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
CitationJournal: Cell Host Microbe / Year: 2023
Title: Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography.
Authors: Pranav N M Shah / James B Gilchrist / Björn O Forsberg / Alister Burt / Andrew Howe / Shyamal Mosalaganti / William Wan / Julika Radecke / Yuriy Chaban / Geoff Sutton / David I Stuart / Mark Boyce /
Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and ...Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.
History
DepositionFeb 24, 2023-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16769.png

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Map

FileDownload / File: emd_16769.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.97 Å/pix.
x 210 pix.
= 413.7 Å		1.97 Å/pix.
x 210 pix.
= 413.7 Å		1.97 Å/pix.
x 210 pix.
= 413.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00713
Minimum - Maximum-0.012813422 - 0.02563859
Average (Standard dev.)0.00012928358 (±0.002585633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 413.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half1

Fileemd_16769_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_16769_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rotavirus A

EntireName: Rotavirus A
Components
  • Virus: Rotavirus A

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Supramolecule #1: Rotavirus A

SupramoleculeName: Rotavirus A / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 28875 / Sci species name: Rotavirus A / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 2.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.0.9) / Number subtomograms used: 412
ExtractionNumber tomograms: 412 / Number images used: 412 / Software - Name: crYOLO (ver. 1.0.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD

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