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- EMDB-26465: In situ structure of Vesicular stomatitis virus (VSV) -

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Basic information

Entry
Database: EMDB / ID: EMD-26465
TitleIn situ structure of Vesicular stomatitis virus (VSV)
Map dataIn situ structure of VSV M-N by subtomogram averaging
Sample
  • Virus: Vesicular stomatitis Indiana virus strain San Juan
Biological speciesVesicular stomatitis Indiana virus strain San Juan
Methodsubtomogram averaging / cryo EM / Resolution: 7.5 Å
AuthorsSi Z
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1U24GM116792 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Locations and in situ structure of the polymerase complex inside the virion of vesicular stomatitis virus.
Authors: Zhu Si / Kang Zhou / Jun Tsao / Ming Luo / Z Hong Zhou /
Abstract: The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA ...The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.
History
DepositionMar 21, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateMay 11, 2022-
Current statusMay 11, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26465.png

Downloads & links

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Map

FileDownload / File: emd_26465.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure of VSV M-N by subtomogram averaging
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.019
Minimum - Maximum-0.07990079 - 0.11618541
Average (Standard dev.)0.00016503056 (±0.0131346835)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 248.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 1 (VSV M-N)

Fileemd_26465_half_map_1.map
Annotationhalf map 1 (VSV M-N)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 (VSV M-N)

Fileemd_26465_half_map_2.map
Annotationhalf map 2 (VSV M-N)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Vesicular stomatitis Indiana virus strain San Juan

EntireName: Vesicular stomatitis Indiana virus strain San Juan
Components
  • Virus: Vesicular stomatitis Indiana virus strain San Juan

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Supramolecule #1: Vesicular stomatitis Indiana virus strain San Juan

SupramoleculeName: Vesicular stomatitis Indiana virus strain San Juan / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 11285
Sci species name: Vesicular stomatitis Indiana virus strain San Juan
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HeLa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 40 / Number images used: 82991
Final angle assignmentType: OTHER / Details: cross-correlation
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 82991
FSC plot (resolution estimation)

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