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- EMDB-2989: The structure of the COPI coat linkage IV -

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Basic information

Entry
Database: EMDB / ID: EMD-2989
TitleThe structure of the COPI coat linkage IV
Map dataReconstruction of the COPI coat linkage IV
Sample
  • Sample: COPI coat linkage IV on the membrane
  • Protein or peptide: Coat protein 1
  • Protein or peptide: ADP-ribosylation factor 1
KeywordsCOPI / coatomer / coated vesicles
Function / homology
Function and homology information


cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / Synthesis of PIPs at the plasma membrane / protein localization to axon / VxPx cargo-targeting to cilium / Synthesis of PIPs at the Golgi membrane / Intra-Golgi traffic / trans-Golgi Network Vesicle Budding / protein localization to Golgi membrane / Golgi localization ...cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / Synthesis of PIPs at the plasma membrane / protein localization to axon / VxPx cargo-targeting to cilium / Synthesis of PIPs at the Golgi membrane / Intra-Golgi traffic / trans-Golgi Network Vesicle Budding / protein localization to Golgi membrane / Golgi localization / COPI-coated vesicle / pancreatic juice secretion / regulation of Golgi organization / organelle membrane contact site / COPI vesicle coat / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / COPI-mediated anterograde transport / Golgi vesicle transport / positive regulation of mitochondrial fusion / COPI-dependent Golgi-to-ER retrograde traffic / organelle transport along microtubule / regulation of fatty acid metabolic process / establishment of Golgi localization / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane transport / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / pigmentation / Golgi-associated vesicle / positive regulation of mitochondrial fission / endoplasmic reticulum-Golgi intermediate compartment / protein secretion / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / Neutrophil degranulation / adult locomotory behavior / small monomeric GTPase / establishment of localization in cell / macroautophagy / protein kinase C binding / intracellular protein transport / hormone activity / protein transport / growth cone / Golgi membrane / axon / GTPase activity / mRNA binding / neuronal cell body / GTP binding / structural molecule activity / Golgi apparatus / endoplasmic reticulum / extracellular space / nucleoplasm / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Coatomer epsilon subunit / Coatomer, epsilon subunit / Coatomer, epsilon subunit / Coatomer beta subunit (COPB1) / Coatomer beta subunit, C-terminal / Coatomer beta subunit (COPB1) / Coatomer beta subunit, appendage platform domain / Coatomer beta C-terminal region / Coatomer beta subunit appendage platform / Coatomer gamma subunit ...Coatomer epsilon subunit / Coatomer, epsilon subunit / Coatomer, epsilon subunit / Coatomer beta subunit (COPB1) / Coatomer beta subunit, C-terminal / Coatomer beta subunit (COPB1) / Coatomer beta subunit, appendage platform domain / Coatomer beta C-terminal region / Coatomer beta subunit appendage platform / Coatomer gamma subunit / Coatomer, gamma subunit, appendage, Ig-like subdomain / Coatomer gamma subunit / Coatomer subunit gamma, C-terminal / Coatomer, gamma subunit, appendage domain superfamily / Coatomer subunit zeta / Coatomer gamma subunit appendage platform subdomain / Coatomer subunit gamma-1 C-terminal appendage platform / Coatomer delta subunit / Coatomer delta subunit / Coatomer subunit alpha / Coatomer, alpha subunit, C-terminal / Coatomer subunit alpha / : / Coatomer (COPI) alpha subunit C-terminus / Coatomer beta' subunit (COPB2) / Coatomer beta' subunit (COPB2) / Coatomer, WD associated region / : / Coatomer WD associated region / Cytochrome cd1-nitrite reductase-like, haem d1 domain superfamily / ADP-ribosylation factor 1-5 / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Small GTPase superfamily, ARF type / Small GTPase superfamily, ARF type / Clathrin adaptor, appendage, Ig-like subdomain superfamily / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / Longin-like domain superfamily / ARF-like small GTPases; ARF, ADP-ribosylation factor / TBP domain superfamily / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Coatomer subunit beta' / Coatomer subunit epsilon / ADP-ribosylation factor 1 / Coatomer subunit zeta-1 / Coatomer subunit delta / Coatomer subunit alpha / Coatomer subunit beta / Coatomer subunit gamma-1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Saccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 21.0 Å
AuthorsDodonova SO / Diestelkoetter-Bachert P / von Appen A / Hagen WJH / Beck R / Beck M / Wieland F / Briggs JAG
CitationJournal: Science / Year: 2015
Title: VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.
Authors: S O Dodonova / P Diestelkoetter-Bachert / A von Appen / W J H Hagen / R Beck / M Beck / F Wieland / J A G Briggs /
Abstract: Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats ...Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.
History
DepositionApr 28, 2015-
Header (metadata) releaseJun 17, 2015-
Map releaseJul 15, 2015-
UpdateJul 22, 2015-
Current statusJul 22, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a1y
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5a1y
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2989.jpg

Downloads & links

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Map

FileDownload / File: emd_2989.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the COPI coat linkage IV
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.02 Å/pix.
x 200 pix.
= 403.8 Å		2.02 Å/pix.
x 200 pix.
= 403.8 Å		2.02 Å/pix.
x 200 pix.
= 403.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.019 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-5.58476925 - 6.96114254
Average (Standard dev.)0.03345215 (±0.92415434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 403.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0192.0192.019
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z403.800403.800403.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-5.5856.9610.033

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Supplemental data

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Sample components

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Entire : COPI coat linkage IV on the membrane

EntireName: COPI coat linkage IV on the membrane
Components
  • Sample: COPI coat linkage IV on the membrane
  • Protein or peptide: Coat protein 1
  • Protein or peptide: ADP-ribosylation factor 1

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Supramolecule #1000: COPI coat linkage IV on the membrane

SupramoleculeName: COPI coat linkage IV on the membrane / type: sample / ID: 1000
Details: The COPI coated vesicles were formed in an in vitro reconstituted budding reaction, which was plunge-frozen without further purification.
Number unique components: 2

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Macromolecule #1: Coat protein 1

MacromoleculeName: Coat protein 1 / type: protein_or_peptide / ID: 1 / Name.synonym: COPI / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse / Location in cell: Golgi, cytoplasm
Molecular weightTheoretical: 560 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFBDM
SequenceInterPro: Coatomer subunit alpha, Coatomer beta' subunit (COPB2), Coatomer, epsilon subunit, Coatomer beta subunit (COPB1), Coatomer delta subunit, Coatomer gamma subunit, AP complex, mu/sigma subunit

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Macromolecule #2: ADP-ribosylation factor 1

MacromoleculeName: ADP-ribosylation factor 1 / type: protein_or_peptide / ID: 2 / Name.synonym: Arf1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Location in cell: Golgi
Molecular weightTheoretical: 20 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pOW12
SequenceUniProtKB: ADP-ribosylation factor 1 / InterPro: Small GTPase superfamily, ARF type

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 50 mM HEPES, 50 mM KAc, 1mM MgCl2
GridDetails: C-Flat Multihole 3C-50 grids glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Instrument: HOMEMADE PLUNGER
Method: The grids were glow discharged for 1 min at 20 mA. 5 ul of 10 nm fiducial gold was added to 40 ul of reaction mix. The reaction mix with the in vitro formed coated vesicles was applied to a ...Method: The grids were glow discharged for 1 min at 20 mA. 5 ul of 10 nm fiducial gold was added to 40 ul of reaction mix. The reaction mix with the in vitro formed coated vesicles was applied to a grid. The grid was blotted for 12 seconds at room temperature before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 89 K / Max: 91 K / Average: 90 K
Specialist opticsEnergy filter - Name: GATAN GIF 2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateFeb 7, 2014
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 45 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsReconstruction was performed using subtomogram averaging. Subtomogram averaging was carried out using scripts from the TOM (Nickell et al, 2005) and AV3 (Foerster et al, 2005) packages. Subtomograms were extracted from the surface of the vesicles.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: TOM, AV3
Details: The reconstructions were carried out for two independent datasets. Two final maps were used to estimate resolution and were averaged together to provide the final map.
Number subtomograms used: 1205
CTF correctionDetails: phase flipping of individual tilts
Final 3D classificationNumber classes: 1
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3mkr


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsThe crystal structure (chains A and B) was fitted into the EM map using global search option in Chimera software.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-5a1y:
The structure of the COPI coat linkage IV

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