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- EMDB-1460: Near-atomic resolution using electron cryomicroscopy and single-p... -

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Basic information

Entry
Database: EMDB / ID: EMD-1460
TitleNear-atomic resolution using electron cryomicroscopy and single-particle reconstruction.
Map dataMap of rotavirus DLP, filtered at 3.8 Angstrom resolution (cosine edge mask), and sharpened with a B-factor of -450 Angstrom squared. The deposited file contains 1/8th of the entire volume.
Sample
  • Sample: Bovine rotavirus DLP
  • Protein or peptide: x 4 types
  • RNA: x 11 types
Biological speciesBovine rotavirus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 5.1 Å
AuthorsZhang X / Settembre E / Xu C / Dormitzer PR
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: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1460.gif

Downloads & links

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Map

FileDownload / File: emd_1460.map.gz / Format: CCP4 / Size: 116.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of rotavirus DLP, filtered at 3.8 Angstrom resolution (cosine edge mask), and sharpened with a B-factor of -450 Angstrom squared. The deposited file contains 1/8th of the entire volume.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 315 pix.
= 779.94 Å		1.24 Å/pix.
x 315 pix.
= 779.94 Å		1.24 Å/pix.
x 315 pix.
= 779.94 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.238 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.111 / Movie #1: 0.06
Minimum - Maximum-0.237517 - 0.301381
Average (Standard dev.)-0.000559727 (±0.0323211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions315315315
Spacing315315315
CellA=B=C: 779.94 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2381.2381.238
M x/y/z630630630
origin x/y/z0.0000.0000.000
length x/y/z779.940779.940779.940
α/β/γ90.00090.00090.000
start NX/NY/NZ-60-60-59
NX/NY/NZ120120120
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS315315315
D min/max/mean-0.2380.301-0.001

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Supplemental data

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Sample components

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Entire : Bovine rotavirus DLP

EntireName: Bovine rotavirus DLP
Components
  • Sample: Bovine rotavirus DLP
  • Protein or peptide: VP1
  • Protein or peptide: VP2
  • Protein or peptide: VP3
  • Protein or peptide: VP6
  • RNA: dsRNA 1RNA
  • RNA: dsRNA 2RNA
  • RNA: dsRNA 3RNA
  • RNA: dsRNA 4RNA
  • RNA: dsRNA 5RNA
  • RNA: dsRNA 6RNA
  • RNA: dsRNA 7RNA
  • RNA: dsRNA 8RNA
  • RNA: dsRNA 9RNA
  • RNA: dsRNA 10RNA
  • RNA: dsRNA 11RNA

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Supramolecule #1000: Bovine rotavirus DLP

SupramoleculeName: Bovine rotavirus DLP / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: 12 monomers of VP1, 120 monomers of VP2, 12
Number unique components: 15
Molecular weightTheoretical: 58 MDa

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Macromolecule #1: VP1

MacromoleculeName: VP1 / type: protein_or_peptide / ID: 1 / Name.synonym: RNA-dependent RNA polymerase / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 125 KDa

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Macromolecule #2: VP2

MacromoleculeName: VP2 / type: protein_or_peptide / ID: 2 / Name.synonym: Virus protein 2 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 41 KDa

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Macromolecule #3: VP3

MacromoleculeName: VP3 / type: protein_or_peptide / ID: 3 / Name.synonym: guanylyltransferase and methylase / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 88 KDa

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Macromolecule #4: VP6

MacromoleculeName: VP6 / type: protein_or_peptide / ID: 4 / Name.synonym: Virus protein 6 / Recombinant expression: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 102 KDa

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Macromolecule #5: dsRNA 1

MacromoleculeName: dsRNA 1 / type: rna / ID: 5 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 2.147 MDa

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Macromolecule #6: dsRNA 2

MacromoleculeName: dsRNA 2 / type: rna / ID: 6 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 1.749 MDa

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Macromolecule #7: dsRNA 3

MacromoleculeName: dsRNA 3 / type: rna / ID: 7 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 1.684 MDa

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Macromolecule #8: dsRNA 4

MacromoleculeName: dsRNA 4 / type: rna / ID: 8 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 1.535 MDa

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Macromolecule #9: dsRNA 5

MacromoleculeName: dsRNA 5 / type: rna / ID: 9 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 1.047 MDa

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Macromolecule #10: dsRNA 6

MacromoleculeName: dsRNA 6 / type: rna / ID: 10 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 881 KDa

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Macromolecule #11: dsRNA 7

MacromoleculeName: dsRNA 7 / type: rna / ID: 11 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 718 KDa

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Macromolecule #12: dsRNA 8

MacromoleculeName: dsRNA 8 / type: rna / ID: 12 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 688 KDa

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Macromolecule #13: dsRNA 9

MacromoleculeName: dsRNA 9 / type: rna / ID: 13 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 690 KDa

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Macromolecule #14: dsRNA 10

MacromoleculeName: dsRNA 10 / type: rna / ID: 14 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 488 KDa

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Macromolecule #15: dsRNA 11

MacromoleculeName: dsRNA 11 / type: rna / ID: 15 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bovine rotavirus
Molecular weightExperimental: 434 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-falt
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: 5.1 Å / Resolution method: OTHER / Software - Name: FREALIGN / Details: Ewald sphere curvature was not corrected / Number images used: 8400
DetailsParticles were selected using the computer program SIGNATURE

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