|Entry||Database: EMDB / ID: 5485|
|Title||Bovine rotavirus DLP|
|Map data||Icosahedral reconstruction of rotavirus double-layered particle using the DE-12 direct electron detector without movie frame alignment|
|Sample||Rotavirus double-layered particle:|
|Keywords||virus / assembly intermediate|
|Method||single particle reconstruction / cryo EM / 7.5 Å resolution|
|Authors||Campbell MG / Cheng A / Brilot AF / Moeller A / Lyumkis D / Veesler D / Pan J / Harrison SC / Potter CS / Carragher B / Grigorieff N|
|Citation||Journal: 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.
|Date||Deposition: Aug 24, 2012 / Header (metadata) release: Sep 12, 2012 / Map release: Sep 12, 2012 / Last update: Nov 21, 2012|
|Structure viewer||EM map: |
Downloads & links
|File||emd_5485.map.gz (map file in CCP4 format, 2000001 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.424 Å|
CCP4 map header:
-Entire Rotavirus double-layered particle
|Entire||Name: Rotavirus double-layered particle / Details: The sample was monodisperse. / Number of components: 1|
Oligomeric State: 12 x VP1, 120 x VP2, 12 x VP3, 780 x VP6, 11 x RNA
|Mass||Theoretical: 70 MDa|
-Component #1: virus, Bovine rotavirus
|Virus||Name: Bovine rotavirus / Class: VIRUS-LIKE PARTICLE / Empty: No / Enveloped: No / Isolate: STRAIN|
|Mass||Theoretical: 70 MDa|
|Species||Species: Bovine rotavirus|
|Source (natural)||Host Species: Bos primigenius / Host category: VERTEBRATES|
|Shell #1||Name of element: DLP / Diameter: 700 Å / T number(triangulation number): 13|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 5 mg/ml / pH: 7.4|
|Support film||1.2-1.3 C-flat|
|Vitrification||Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 85 % / Method: Blot for 7 seconds before plunging|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Nov 28, 2011|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 42135 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2400 nm|
|Specimen Holder||Model: GATAN LIQUID NITROGEN|
|Camera||Detector: DIRECT ELECTRON DE-12 (4k x 3k)|
|Image acquisition||Number of digital images: 501 / Sampling size: 6 microns / Details: Movies recorded at 25 frames/second|
|Processing||Method: single particle reconstruction|
Details: Manual particle selection, refinement using Frealign.
Number of projections: 807 / Applied symmetry: I (icosahedral)
|3D reconstruction||Algorithm: Projection matching / Software: Frealign / CTF correction: Each particle|
Details: Final map was calculated from 807 particles with icosahedral symmetry imposed
Resolution: 7.5 Å / Resolution method: FSC 0.143
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