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- EMDB-3720: The structure of the COPI coat leaf -

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Basic information

Entry
Database: EMDB / ID: EMD-3720
TitleThe structure of the COPI coat leaf
Map data
SampleThe structure of the COPI coat leaf
  • COPI coat complex
  • (ADP-ribosylation factor ...ADP ribosylation factor) x 2
  • (Coatomer subunit ...) x 6
Function / homology
Function and homology information


cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / protein localization to axon / Golgi localization / pancreatic juice secretion / COPI vesicle coat / Golgi vesicle transport / organelle transport along microtubule / COPI-coated vesicle / intra-Golgi vesicle-mediated transport ...cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / protein localization to axon / Golgi localization / pancreatic juice secretion / COPI vesicle coat / Golgi vesicle transport / organelle transport along microtubule / COPI-coated vesicle / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane transport / pigmentation / establishment of Golgi localization / Golgi-associated vesicle / toxin transport / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / protein secretion / endoplasmic reticulum-Golgi intermediate compartment / adult locomotory behavior / vesicle-mediated transport / protein kinase C binding / intracellular protein transport / macroautophagy / hormone activity / cell body / protein transport / growth cone / endoplasmic reticulum to Golgi vesicle-mediated transport / GTPase activity / Golgi membrane / mRNA binding / axon / intracellular membrane-bounded organelle / GTP binding / structural molecule activity / Golgi apparatus / endoplasmic reticulum / extracellular space / membrane / plasma membrane / cytosol / cytoplasm
Clathrin adaptor complex, small chain / Cytochrome cd1-nitrite reductase-like, haem d1 domain superfamily / G-protein beta WD-40 repeat / WD40 repeat / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Small GTP-binding protein domain / Small GTPase superfamily, ARF/SAR type / Coatomer, WD associated region / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Coatomer, alpha subunit, C-terminal ...Clathrin adaptor complex, small chain / Cytochrome cd1-nitrite reductase-like, haem d1 domain superfamily / G-protein beta WD-40 repeat / WD40 repeat / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Small GTP-binding protein domain / Small GTPase superfamily, ARF/SAR type / Coatomer, WD associated region / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Coatomer, alpha subunit, C-terminal / Longin-like domain superfamily / Coatomer beta subunit, C-terminal / Anaphase-promoting complex subunit 4, WD40 domain / Armadillo-like helical / TBP domain superfamily / Coatomer, gamma subunit, appendage, Ig-like subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Armadillo-type fold / Coatomer alpha subunit / Coatomer beta' subunit (COPB2) / Coatomer beta subunit (COPB1) / Coatomer gamma subunit / WD40 repeat, conserved site / AP complex, mu/sigma subunit / WD40-repeat-containing domain / Coatomer beta subunit, appendage platform domain / Mu homology domain / P-loop containing nucleoside triphosphate hydrolase / Coatomer subunit gamma, C-terminal / Coatomer subunit zeta / Coatomer, gamma subunit, appendage domain superfamily / Coatomer delta subunit / WD40-repeat-containing domain superfamily / AP-2 complex subunit mu, C-terminal superfamily
Coatomer subunit beta' / ADP-ribosylation factor 1 / Coatomer subunit zeta-1 / Coatomer subunit delta / Coatomer subunit alpha / Coatomer subunit beta / Coatomer subunit gamma-1
Biological speciesMus musculus (house mouse) / Saccharomyces cerevisiae (baker's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 9.2 Å
AuthorsDodonova SO / Aderhold P / Kopp J / Ganeva I / Roehling S / Hagen WJH / Sinning I / Wieland F / Briggs JAG
CitationJournal: Elife / Year: 2017
Title: 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.
Authors: Svetlana O Dodonova / Patrick Aderhold / Juergen Kopp / Iva Ganeva / Simone Röhling / Wim J H Hagen / Irmgard Sinning / Felix Wieland / John A G Briggs /
Abstract: COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that ...COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 15, 2017-
Header (metadata) releaseMay 31, 2017-
Map releaseJun 28, 2017-
UpdateOct 24, 2018-
Current statusOct 24, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5nzr
  • Surface level: 0.12
  • 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_3720.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.78 Å/pix.
x 128 pix.
= 227.84 Å
1.78 Å/pix.
x 128 pix.
= 227.84 Å
1.78 Å/pix.
x 128 pix.
= 227.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.78 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.40815967 - 0.55549365
Average (Standard dev.)-0.000038523292 (±0.05965102)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 227.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z227.840227.840227.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.4080.555-0.000

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Supplemental data

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Sample components

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Entire The structure of the COPI coat leaf

EntireName: The structure of the COPI coat leaf / Details: The asymmetric unit of the COPI coat / Number of components: 10

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Component #1: protein, The structure of the COPI coat leaf

ProteinName: The structure of the COPI coat leaf / Details: The asymmetric unit of the COPI coat / Recombinant expression: No
MassTheoretical: 550 kDa

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Component #2: protein, COPI coat complex

ProteinName: COPI coat complex / Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, ADP-ribosylation factor 1

ProteinName: ADP-ribosylation factor 1ARF1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Coatomer subunit alpha

ProteinName: Coatomer subunit alpha / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 142.53275 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, Coatomer subunit beta

ProteinName: Coatomer subunit beta / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 109.148109 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: protein, Coatomer subunit beta'

ProteinName: Coatomer subunit beta' / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 102.566078 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #7: protein, Coatomer subunit delta

ProteinName: Coatomer subunit delta / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 57.30425 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #8: protein, ADP-ribosylation factor 1

ProteinName: ADP-ribosylation factor 1ARF1 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 20.552438 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #9: protein, Coatomer subunit gamma-1

ProteinName: Coatomer subunit gamma-1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 97.622703 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #10: protein, Coatomer subunit zeta-1

ProteinName: Coatomer subunit zeta-1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 20.218168 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Sample preparation

SpecimenSpecimen state: 3D array / Method: cryo EM
Sample solutionBuffer solution: Protein-A conjugated 10 nm gold was added to the reaction mix in 1:6 volume ratio before plunge-freezing
pH: 7.4
Support filmProtochips C-flat MultiHole, 20 mA
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 296 K / Humidity: 85 %
Details: The sample was applied onto glow-discharged (30 sec, 20 mA) C-flat (Protochips Inc.) multihole grids. The grids were blotted from the back side for 11 seconds at room temperature in a ...Details: The sample was applied onto glow-discharged (30 sec, 20 mA) C-flat (Protochips Inc.) multihole grids. The grids were blotted from the back side for 11 seconds at room temperature in a chamber at 85% humidity and plunge-frozen into liquid ethane using a manual plunger..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2500.0 - 5000.0 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionDetails: Each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015).

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 38498
3D reconstructionSoftware: TOM Toolbox, AV3
CTF correction: CTF-determination for each individual tilt image was performed using CTFFIND4. Strip-based CTF-correction and tomogram reconstruction was performed in Imod.
Resolution: 9.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Cross-correlation coefficient / Refinement space: REAL
Output model

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