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- EMDB-5269: 3D reconstruction of yeast coatomer -

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Basic information

Entry
Database: EMDB / ID: EMD-5269
Title3D reconstruction of yeast coatomer
Map data3D reconstruction of yeast coatomer
Sample
  • Sample: Yeast coatomer complex
  • Protein or peptide: Cop1
  • Protein or peptide: Sec26
  • Protein or peptide: Sec27
  • Protein or peptide: Sec21
  • Protein or peptide: Ret2
  • Protein or peptide: Sec28
  • Protein or peptide: Ret3
Keywordscoatomer / COP1 vesicle / vesicular trafficking / coat complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 36.0 Å
AuthorsYip CK / Walz T
CitationJournal: J Mol Biol / Year: 2011
Title: Molecular structure and flexibility of the yeast coatomer as revealed by electron microscopy.
Authors: Calvin K Yip / Thomas Walz /
Abstract: Coat protein complex I (COPI)-coated vesicles, one of three major types of vesicular carriers in the cell, mediate the early secretory pathway and retrograde transport from the Golgi to the ...Coat protein complex I (COPI)-coated vesicles, one of three major types of vesicular carriers in the cell, mediate the early secretory pathway and retrograde transport from the Golgi to the endoplasmic reticulum. COPI vesicles are generated through activation of the regulatory GTPase Arf1 at the donor membrane and the subsequent recruitment of coatomer, a coat protein complex consisting of seven stably associated components. Coatomer functions in binding and sequestering cargo molecules and assembles into a polymeric protein shell that encompasses the surface of COPI vesicles. Little is known about the structural properties of this heptameric complex. We have isolated native yeast coatomer and examined its structure and subunit organization by single-particle electron microscopy. Our analyses provide the first three-dimensional picture of the complete coatomer and reveal substantial conformational flexibility likely to be critical for its scaffolding function.
History
DepositionMar 18, 2011-
Header (metadata) releaseMar 21, 2011-
Map releaseMar 29, 2011-
UpdateSep 23, 2011-
Current statusSep 23, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5269.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of yeast coatomer
Voxel sizeX=Y=Z: 4.2 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.0210324 - 0.488452
Average (Standard dev.)0.00491068 (±0.0343284)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 378 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-0.0210.4880.005

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Supplemental data

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Sample components

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Entire : Yeast coatomer complex

EntireName: Yeast coatomer complex
Components
  • Sample: Yeast coatomer complex
  • Protein or peptide: Cop1
  • Protein or peptide: Sec26
  • Protein or peptide: Sec27
  • Protein or peptide: Sec21
  • Protein or peptide: Ret2
  • Protein or peptide: Sec28
  • Protein or peptide: Ret3

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Supramolecule #1000: Yeast coatomer complex

SupramoleculeName: Yeast coatomer complex / type: sample / ID: 1000 / Oligomeric state: one heteroheptamer / Number unique components: 7
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: Cop1

MacromoleculeName: Cop1 / type: protein_or_peptide / ID: 1 / Name.synonym: alpha / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 136 KDa

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Macromolecule #2: Sec26

MacromoleculeName: Sec26 / type: protein_or_peptide / ID: 2 / Name.synonym: beta / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 109 KDa

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Macromolecule #3: Sec27

MacromoleculeName: Sec27 / type: protein_or_peptide / ID: 3 / Name.synonym: beta' / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 109 KDa

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Macromolecule #4: Sec21

MacromoleculeName: Sec21 / type: protein_or_peptide / ID: 4 / Name.synonym: gamma / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 105 KDa

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Macromolecule #5: Ret2

MacromoleculeName: Ret2 / type: protein_or_peptide / ID: 5 / Name.synonym: delta / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 61 KDa

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Macromolecule #6: Sec28

MacromoleculeName: Sec28 / type: protein_or_peptide / ID: 6 / Name.synonym: epsilon / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 34 KDa

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Macromolecule #7: Ret3

MacromoleculeName: Ret3 / type: protein_or_peptide / ID: 7 / Name.synonym: zeta / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Cell: yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 22 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

Concentration0.02 mg/mL
BufferpH: 8
Details: 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 0.005% NP40, 0.5 mM DTT
StainingType: NEGATIVE
Details: Protein solution was adsorbed onto grids for 30 seconds before staining with 0.75% uranyl formate
VitrificationCryogen name: NITROGEN / Chamber temperature: 100 K / Instrument: OTHER / Method: Manual plunging into liquid nitrogen

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51159 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.4 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: OTHER / Tilt angle max: 50
TemperatureAverage: 100 K
DetailsLow dose imaging, same specimen area imaged twice
DateMar 1, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final angle assignmentDetails: SPIDER:theta 45 degrees, phi 45 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 36.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: Final map is filtered to 36 Angstroms resolution

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