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- EMDB-1461: Near-atomic resolution using electron cryomicroscopy and single-p... -

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Basic information

Entry
Database: EMDB / ID: EMD-1461
TitleNear-atomic resolution using electron cryomicroscopy and single-particle reconstruction.
Map dataMap of rotavirus VP6 protein, after icosahedral averaging and 13-fold non-icosahedral averaging
Sample
  • Sample: Rotavirus VP6 protein
  • Protein or peptide: VP6
Biological speciesBovine rotavirus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 3.8 Å
AuthorsZhang X / Settembre E / Xu C / Dormitzer PR / Bellamy R / Harrison SC / Grigorieff N
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction.
Authors: Xing Zhang / Ethan Settembre / Chen Xu / Philip R Dormitzer / Richard Bellamy / Stephen C Harrison / Nikolaus Grigorieff /
Abstract: Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as ...Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as "single-particle reconstruction." During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle ("double-layer particle" or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a high-resolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-A resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.
History
DepositionDec 10, 2007-
Header (metadata) releaseDec 10, 2007-
Map releaseJan 2, 2008-
UpdateOct 31, 2012-
Current statusOct 31, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1qhd
  • Surface level: 0.775444175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1461.gif

Downloads & links

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Map

FileDownload / File: emd_1461.map.gz / Format: CCP4 / Size: 6.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of rotavirus VP6 protein, after icosahedral averaging and 13-fold non-icosahedral averaging
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.23 Å/pix.
x 134 pix.
= 774.9 Å		1.23 Å/pix.
x 106 pix.
= 774.9 Å		1.23 Å/pix.
x 122 pix.
= 774.9 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.04 / Movie #1: 1
Minimum - Maximum-2.16499 - 3.11145
Average (Standard dev.)0.0102024 (±0.246157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin29-50166
Dimensions106122134
Spacing106122134
CellA=B=C: 774.9 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z630630630
origin x/y/z0.0000.0000.000
length x/y/z774.900774.900774.900
α/β/γ90.00090.00090.000
start NX/NY/NZ29-50166
NX/NY/NZ106122134
MAP C/R/S213
start NC/NR/NS-5029166
NC/NR/NS122106134
D min/max/mean-2.1653.1110.010

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Supplemental data

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Sample components

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Entire : Rotavirus VP6 protein

EntireName: Rotavirus VP6 protein
Components
  • Sample: Rotavirus VP6 protein
  • Protein or peptide: VP6

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Supramolecule #1000: Rotavirus VP6 protein

SupramoleculeName: Rotavirus VP6 protein / type: sample / ID: 1000
Details: VP6 is one component of rotavirus DLP, which has been deposited separately (accession number EMD-1460)
Oligomeric state: 780 molecules of VP6 form a DLP particle with 12 molecules of VP1, 120 molecules of VP2, 12 molecules of VP3 and 11 dsRNA molecules
Number unique components: 1
Molecular weightTheoretical: 41 KDa

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Macromolecule #1: VP6

MacromoleculeName: VP6 / type: protein_or_peptide / ID: 1 / Name.synonym: Virus protein 6 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 41 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Details: Ice
GridDetails: Lacy carbon and C-flat
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Home-made. Vitrification carried out in air at room temperature
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 56540 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Eucentric, side-entry / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected
Legacy - Electron beam tilt params: 0
DateJun 1, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 386 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: FREALIGN
Details: The FREALIGN reconstruction was followed by non-icosahedral averaging using the Uppsala program package for X-ray crystallography
Number images used: 8400
DetailsParticles were selected using the computer program SIGNATURE

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