+Open data
-Basic information
Entry | Database: PDB / ID: 5nzv | |||||||||
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Title | The structure of the COPI coat linkage IV | |||||||||
Components |
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Keywords | TRANSPORT PROTEIN / COPI / coatomer / coated vesicles | |||||||||
Function / homology | Function and homology information cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / protein localization to axon / VxPx cargo-targeting to cilium / Intra-Golgi traffic / Synthesis of PIPs at the Golgi membrane / trans-Golgi Network Vesicle Budding / Golgi localization / COPI-coated vesicle / pancreatic juice secretion ...cerebellar Purkinje cell layer maturation / protein localization to cell leading edge / protein localization to axon / VxPx cargo-targeting to cilium / Intra-Golgi traffic / Synthesis of PIPs at the Golgi membrane / trans-Golgi Network Vesicle Budding / Golgi localization / COPI-coated vesicle / pancreatic juice secretion / COPI vesicle coat / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / COPI-mediated anterograde transport / Golgi vesicle transport / COPI-dependent Golgi-to-ER retrograde traffic / organelle transport along microtubule / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane transport / establishment of Golgi localization / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / pigmentation / Golgi-associated vesicle / protein secretion / endoplasmic reticulum-Golgi intermediate compartment / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / Neutrophil degranulation / adult locomotory behavior / small monomeric GTPase / establishment of localization in cell / protein kinase C binding / macroautophagy / intracellular protein transport / hormone activity / protein transport / growth cone / axon / Golgi membrane / intracellular membrane-bounded organelle / GTPase activity / mRNA binding / neuronal cell body / structural molecule activity / GTP binding / Golgi apparatus / endoplasmic reticulum / extracellular space / nucleoplasm / plasma membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | ELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 17.3 Å | |||||||||
Authors | Dodonova, 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
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5nzv.cif.gz | 1.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb5nzv.ent.gz | 781.7 KB | Display | PDB format |
PDBx/mmJSON format | 5nzv.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nz/5nzv ftp://data.pdbj.org/pub/pdb/validation_reports/nz/5nzv | HTTPS FTP |
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-Related structure data
Related structure data | 3724MC 3720C 3721C 3722C 3723C 5mu7C 5nzrC 5nzsC 5nztC 5nzuC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Coatomer subunit ... , 7 types, 17 molecules AHEOBICJDNGKQZLUS
#1: Protein | Mass: 142532.750 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copa / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8CIE6 #2: Protein | Mass: 34605.055 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cope, Cope1 / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O89079 #3: Protein | Mass: 109148.109 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copb1, Copb / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9JIF7 #4: Protein | Mass: 102566.078 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copb2 / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O55029 #5: Protein | Mass: 57304.250 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Arcn1, Copd / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q5XJY5 #7: Protein | Mass: 97622.703 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copg1, Copg / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9QZE5 #8: Protein | Mass: 20218.168 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Copz1, Copz / Plasmid: pFBDM / Cell line (production host): SF9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P61924 |
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-Protein , 1 types, 5 molecules FRMPT
#6: Protein | Mass: 20552.438 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: ARF1, YDL192W, D1244 / Plasmid: pOW12 / Production host: Escherichia coli (E. coli) / References: UniProt: P11076 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: subtomogram averaging |
-Sample preparation
Component |
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Molecular weight | Value: 2.2 MDa / Experimental value: NO | ||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 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 |
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Specimen | Embedding 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 support | Details: Protochips C-flat MultiHole 20 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat | ||||||||||||||||||||||||||||
Vitrification | Instrument: 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS Details: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017) |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5000 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron 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 optics | Energyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV |
Image scans | Movie frames/image: 5 / Used frames/image: 1-5 |
-Processing
EM software |
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CTF correction | Details: 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 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 17.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1640 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
EM volume selection | Details: 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: 1640 | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient |