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Yorodumi- EMDB-3973: The in situ structure of the Chlamydomonas COPI coat: average fro... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3973 | |||||||||
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Title | The in situ structure of the Chlamydomonas COPI coat: average from the trans-Golgi/TGN region | |||||||||
Map data | The in situ structure of the Chlamydomonas COPI coat derived from the trans-Golgi/TGN region. | |||||||||
Sample |
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Biological species | Chlamydomonas reinhardtii (plant) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 24.0 Å | |||||||||
Authors | Bykov YS / Schaffer M / Dodonova SO / Albert S / Plitzko JM / Baumeister W / Engel BD / Briggs JAG | |||||||||
Citation | Journal: Elife / Year: 2017 Title: The structure of the COPI coat determined within the cell. Authors: Yury S Bykov / Miroslava Schaffer / Svetlana O Dodonova / Sahradha Albert / Jürgen M Plitzko / Wolfgang Baumeister / Benjamin D Engel / John Ag Briggs / Abstract: COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively ...COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with reconstitution systems using purified components. Previously we have determined a complete structural model of the reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified cells. The native algal structure resembles the mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis , maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from to , but the structure of the coat machinery remains constant. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3973.map.gz | 7.3 MB | EMDB map data format | |
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Header (meta data) | emd-3973-v30.xml emd-3973.xml | 11.6 KB 11.6 KB | Display Display | EMDB header |
Images | emd_3973.png | 92.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3973 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3973 | HTTPS FTP |
-Validation report
Summary document | emd_3973_validation.pdf.gz | 233.1 KB | Display | EMDB validaton report |
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Full document | emd_3973_full_validation.pdf.gz | 232.2 KB | Display | |
Data in XML | emd_3973_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3973 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3973 | HTTPS FTP |
-Related structure data
Related structure data | 3968C 3969C 3970C 3971C 3972C 3974C 3975C 3976C 3977C C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3973.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | The in situ structure of the Chlamydomonas COPI coat derived from the trans-Golgi/TGN region. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.42 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Whole Chlamydomonas cells
Entire | Name: Whole Chlamydomonas cells |
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Components |
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-Supramolecule #1: Whole Chlamydomonas cells
Supramolecule | Name: Whole Chlamydomonas cells / type: organelle_or_cellular_component / ID: 1 / Parent: 0 Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light |
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Source (natural) | Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4 / Organelle: Golgi |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7 / Details: TAP media |
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV Details: Blotted from the back side for 10 seconds with 10 blot force before plunging. |
Details | The cells were frozen onto grids, then thinned using cryo-focused ion beam milling. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-12 / Average exposure time: 1.5 sec. / Average electron dose: 1.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: TOM, AV3) Details: Only subtomograms contained in buds and vesicles of the trans-Golgi/TGN region were used to generate this average. No additional alignment was performed. Number subtomograms used: 630 |
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Extraction | Number tomograms: 29 / Number images used: 53109 / Reference model: None / Software: (Name: TOM, AV3) Details: Subtomograms were extracted uniformly around the membrane surface with initial angles normal to it. |
CTF correction | Software: (Name: CTFFIND, CTFPHASEFLIP) |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: AV3 |
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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