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- PDB-5nzu: The structure of the COPI coat linkage II -

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Basic information

Entry
Database: PDB / ID: 5nzu
TitleThe structure of the COPI coat linkage II
Components
  • (Coatomer subunit ...) x 6
  • ADP-ribosylation factor 1
KeywordsTRANSPORT PROTEIN / COPI / 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 / plasma membrane / cytosol / cytoplasm
Similarity search - Function
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, appendage, Ig-like subdomain / Coatomer gamma subunit / Coatomer subunit gamma, C-terminal / Coatomer, gamma subunit, appendage domain superfamily / Coatomer subunit zeta ...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, 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, alpha subunit, C-terminal / Coatomer subunit alpha / : / Coatomer (COPI) alpha subunit C-terminus / 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 / 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 / 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 / Anaphase-promoting complex subunit 4 WD40 domain / ARF-like small GTPases; ARF, ADP-ribosylation factor / TBP domain superfamily / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / 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' / 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)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 15 Å
AuthorsDodonova, S.O. / Aderhold, P. / Kopp, J. / Ganeva, I. / Roehling, S. / Hagen, W.J.H. / Sinning, I. / Wieland, F. / Briggs, J.A.G.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research FoundationSFB638 (A16 and Z4) Germany
German Research FoundationWI 654/12-1 Germany
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.
History
DepositionMay 15, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Experimental preparation / Refinement description
Category: em_3d_fitting / em_imaging_optics ...em_3d_fitting / em_imaging_optics / em_sample_support / em_software / pdbx_audit_support
Item: _em_3d_fitting.target_criteria / _em_imaging_optics.energyfilter_name ..._em_3d_fitting.target_criteria / _em_imaging_optics.energyfilter_name / _em_sample_support.details / _em_sample_support.grid_type / _pdbx_audit_support.funding_organization
Revision 1.2Jan 31, 2018Group: Data processing / Category: em_software / Item: _em_software.name / _em_software.version
Revision 1.3Oct 24, 2018Group: Advisory / Data collection / Derived calculations / Category: pdbx_validate_close_contact / struct_conn
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Coatomer subunit alpha
B: Coatomer subunit beta
C: Coatomer subunit beta'
D: Coatomer subunit delta
F: ADP-ribosylation factor 1
G: Coatomer subunit gamma-1
R: ADP-ribosylation factor 1
Z: Coatomer subunit zeta-1
K: Coatomer subunit gamma-1
L: Coatomer subunit zeta-1
M: ADP-ribosylation factor 1


Theoretical massNumber of molelcules
Total (without water)708,89011
Polymers708,89011
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models3

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Components

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Coatomer subunit ... , 6 types, 8 molecules ABCDGKZL

#1: Protein Coatomer subunit alpha / Alpha-coat protein / Alpha-COP


Mass: 142532.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copa / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8CIE6
#2: Protein Coatomer subunit beta / Beta-coat protein / Beta-COP


Mass: 109148.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copb1, Copb / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9JIF7
#3: Protein Coatomer subunit beta' / Beta'-coat protein / Beta'-COP / p102


Mass: 102566.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copb2 / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O55029
#4: Protein Coatomer subunit delta / Archain / Delta-coat protein / Delta-COP


Mass: 57304.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Arcn1, Copd / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q5XJY5
#6: Protein Coatomer subunit gamma-1 / Gamma-1-coat protein / Gamma-1-COP


Mass: 97622.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copg1, Copg / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9QZE5
#7: Protein Coatomer subunit zeta-1 / Zeta-1-coat protein / Zeta-1 COP


Mass: 20218.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copz1, Copz / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P61924

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Protein , 1 types, 3 molecules FRM

#5: Protein ADP-ribosylation factor 1


Mass: 20552.438 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ARF1, YDL192W, D1244 / Production host: Escherichia coli (E. coli) / References: UniProt: P11076

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1The structure of the COPI coat leafCOMPLEXThe asymmetric unit of the COPI coatall0MULTIPLE SOURCES
2COPI coat complexCOMPLEX#1-#4, #6-#71RECOMBINANT
3ADP-ribosylation factor 1COMPLEX#51RECOMBINANT
Molecular weightValue: 1.65 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Details: Protein-A conjugated 10 nm gold was added to the reaction mix in 1:6 volume ratio before plunge-freezing
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
250 mMKOAc1
31 mMMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: COPI-coated vesicles were produced in vitro by incubating coatomer, Arf1, GTPgS, ARNO and GUVs in a total volume of 40 ul for 30 minutes at 37C
Specimen supportDetails: Protochips C-flat MultiHole, 20 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 296 K
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
MicroscopyModel: 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 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
Details: 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).
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 5 / Used frames/image: 1-5

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Processing

EM software
IDNameVersionCategory
1Amiravolume selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.11model fitting
13TOM Toolbox3D reconstruction
14TOM Toolboxvolume selection
15IMODCTF correction
16AV33D reconstruction
CTF correctionDetails: CTF-determination for each individual tilt image was performed using CTFFIND4. Strip-based CTF-correction and tomogram reconstruction was performed in Imod.
Type: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3312 / Symmetry type: POINT
EM volume selectionDetails: 1733 vesicles and near-complete buds were picked from 61 tomograms. Subtomograms were extracted from the surface of the vesicles.
Num. of tomograms: 54 / Num. of volumes extracted: 3312
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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