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- EMDB-20593: Rotavirus, transcriptionally active -

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Basic information

Entry
Database: EMDB / ID: EMD-20593
TitleRotavirus, transcriptionally active
Map dataRotavirus actively transcribing
Sample
  • Virus: Simian rotavirus A/SA11-4F
Biological speciesSimian rotavirus A/SA11-4F
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 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: 0.0812
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0812
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20593.png

Downloads & links

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Map

FileDownload / File: emd_20593.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRotavirus actively transcribing
Voxel sizeX=Y=Z: 4.4 Å
Density
Contour LevelBy AUTHOR: 0.0812 / Movie #1: 0.0812
Minimum - Maximum-0.016788542 - 0.10787897
Average (Standard dev.)0.019129312 (±0.03691989)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 880.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z880.000880.000880.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0170.1080.019

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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
Components
  • Virus: Simian rotavirus A/SA11-4F

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Supramolecule #1: Simian rotavirus A/SA11-4F

SupramoleculeName: Simian rotavirus A/SA11-4F / type: virus / ID: 1 / Parent: 0
Details: Simian Rotavirus double-layered particle assemblies purified using isopycnic centrifugation in cesium chloride
NCBI-ID: 36436 / Sci species name: Simian rotavirus A/SA11-4F / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Simian rotavirus A/SA11-4F
Virus shellShell ID: 1 / Name: VP6 / Diameter: 800.0 Å

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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

Concentration1 mg/mL
BufferpH: 7.5
Details: 50mM HEPES, pH7.5, 150mM NaCl, 10mM MgCl2 and 10mM CaCl2
GridDetails: Functionalized with Nickle-nitrilotriacetic acid (ni-NTA) monolayers composed of 25% Ni-NTA lipids and 75% 1,2-dilaurylphosphatidylchlorine filler lipids (Avanti Polar lipids)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III / Details: Sample was frozen on C-flat holey carbon grids.
Detailssample was monodispersed

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus min: -1.5 µm / Nominal magnification: 68000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI EAGLE (2k x 2k) / Average electron dose: 5.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 467
CTF correctionSoftware - Name: RELION (ver. 2.0)
Startup modelType of model: OTHER
Details: DLP density map deposited on the Grigorieff website
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 2.0) / Details: RELION software package
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: OTHER / Details: RELION software package
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 467

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