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- EMDB-5486: Bovine rotavirus DLP -

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Basic information

Entry
Database: EMDB / ID: EMD-5486
TitleBovine rotavirus DLP
Map dataIcosahedral reconstruction of rotavirus double-layered particle using the DE-12 direct electron detector with movie frame alignment
Sample
  • Sample: Rotavirus double-layered particle
  • Virus: Bovine rotavirus
Keywordsvirus / assembly intermediate
Biological speciesBovine rotavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsCampbell MG / Cheng A / Brilot AF / Moeller A / Lyumkis D / Veesler D / Pan J / Harrison SC / Potter CS / Carragher B / Grigorieff N
CitationJournal: Structure / Year: 2012
Title: Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.
Authors: Melody G Campbell / Anchi Cheng / Axel F Brilot / Arne Moeller / Dmitry Lyumkis / David Veesler / Junhua Pan / Stephen C Harrison / Clinton S Potter / Bridget Carragher / Nikolaus Grigorieff /
Abstract: Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We ...Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
History
DepositionAug 24, 2012-
Header (metadata) releaseSep 12, 2012-
Map releaseSep 12, 2012-
UpdateNov 21, 2012-
Current statusNov 21, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5486_1.png

Downloads & links

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Map

FileDownload / File: emd_5486.map.gz / Format: CCP4 / Size: 1.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedral reconstruction of rotavirus double-layered particle using the DE-12 direct electron detector with movie frame alignment
Voxel sizeX=Y=Z: 1.424 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.17343959 - 0.28726926
Average (Standard dev.)-0.03809622 (±0.04043201)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions800800800
Spacing800800800
CellA=B=C: 1139.2001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.4241.4241.424
M x/y/z800800800
origin x/y/z0.0000.0000.000
length x/y/z1139.2001139.2001139.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS800800800
D min/max/mean-0.1730.287-0.038

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Supplemental data

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Sample components

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Entire : Rotavirus double-layered particle

EntireName: Rotavirus double-layered particle
Components
  • Sample: Rotavirus double-layered particle
  • Virus: Bovine rotavirus

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Supramolecule #1000: Rotavirus double-layered particle

SupramoleculeName: Rotavirus double-layered particle / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: 12 x VP1, 120 x VP2, 12 x VP3, 780 x VP6, 11 x RNA
Number unique components: 1
Molecular weightTheoretical: 70 MDa

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Supramolecule #1: Bovine rotavirus

SupramoleculeName: Bovine rotavirus / type: virus / ID: 1 / NCBI-ID: 10927 / Sci species name: Bovine rotavirus / Database: NCBI / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Bos primigenius (aurochs) / synonym: VERTEBRATES
Molecular weightTheoretical: 70 MDa
Virus shellShell ID: 1 / Name: DLP / Diameter: 700 Å / T number (triangulation number): 13

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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

Concentration5 mg/mL
BufferpH: 7.4
GridDetails: 1.2-1.3 C-flat
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 7 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42135 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateNov 28, 2011
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Sampling interval: 6 µm / Number real images: 501 / Average electron dose: 32 e/Å2 / Details: Movies recorded at 25 frames/second
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Frealign
Details: Final map was calculated from 807 particles with icosahedral symmetry imposed
Number images used: 807
DetailsManual particle selection, refinement using Frealign.

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