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- EMDB-20592: Rotavirus, transcriptionally inactive -

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Basic information

Entry
Database: EMDB / ID: EMD-20592
TitleRotavirus, transcriptionally inactive
Map data
SampleSimian rotavirus A/SA11-4F:
virus
Biological speciesSimian rotavirus A/SA11-4F
Methodsingle particle reconstruction / cryo EM / Resolution: 10 Å
AuthorsKelly DF / Hauser M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteR01CA193578 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01AI116815 United States
CitationJournal: Comput Struct Biotechnol J / Year: 2019
Title: Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles.
Authors: Mary Hauser / William J Dearnaley / A Cameron Varano / Michael Casasanta / Sarah M McDonald / Deborah F Kelly /
Abstract: Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is ...Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach.
History
DepositionAug 13, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseSep 11, 2019-
UpdateSep 11, 2019-
Current statusSep 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20592.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.4 Å/pix.
x 100 pix.
= 440. Å
4.4 Å/pix.
x 100 pix.
= 440. Å
4.4 Å/pix.
x 100 pix.
= 440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.4 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.05317041 - 0.18961222
Average (Standard dev.)0.0474684 (±0.06571136)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z440.000440.000440.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0530.1900.047

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Supplemental data

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Sample components

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Entire Simian rotavirus A/SA11-4F

EntireName: Simian rotavirus A/SA11-4F
Details: Simian Rotavirus double-layered particle assemblies purified using isopycnic centrifugation in cesium chloride
Number of components: 1

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Component #1: virus, Simian rotavirus A/SA11-4F

VirusName: Simian rotavirus A/SA11-4F / Class: VIRION
Details: Simian Rotavirus double-layered particle assemblies purified using isopycnic centrifugation in cesium chloride
Empty: No / Enveloped: No / Isolate: STRAIN
SpeciesSpecies: Simian rotavirus A/SA11-4F
Source (natural)Host Species: Simian rotavirus A/SA11-4F
Shell #1Name of element: VP6 / Diameter: 800.0 Å

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL
Buffer solution: 50mM HEPES, pH7.5, 150mM NaCl, 10mM MgCl2 and 10mM CaCl2
pH: 7.5
Support filmFunctionalized with Nickel-nitrilotriacetic acide (Ni-NTA) monolayers composed of 25% Ni-NTA lipids and 75% 1,2-dilaurylphosphatidylchlorine filler lipids (Avanti Polar lipids).
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 90 % / Details: Sample was frozen on C-flat holey carbon grids.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI SPIRIT
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 68000.0 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: -1500.0 - nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: FEI EAGLE (2k x 2k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 452
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 10 Å / Resolution method: FSC 0.5 CUT-OFF / Euler angles: RELION software package

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