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- EMDB-3976: The in situ structure of the Chlamydomonas COPI coat linkage IV -

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Basic information

Entry
Database: EMDB / ID: EMD-3976
TitleThe in situ structure of the Chlamydomonas COPI coat linkage IV
Map dataThe in situ structure of the Chlamydomonas COPI coat linkage IV
Sample
  • Organelle or cellular component: Whole Chlamydomonas cells
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 32.0 Å
AuthorsBykov YS / Schaffer M / Dodonova SO / Albert S / Plitzko JM / Baumeister W / Engel BD / Briggs JAG
CitationJournal: 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
DepositionNov 7, 2017-
Header (metadata) releaseNov 15, 2017-
Map releaseNov 29, 2017-
UpdateNov 29, 2017-
Current statusNov 29, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3976.png

Downloads & links

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Map

FileDownload / File: emd_3976.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe in situ structure of the Chlamydomonas COPI coat linkage IV
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.84 Å/pix.
x 128 pix.
= 875.52 Å		6.84 Å/pix.
x 128 pix.
= 875.52 Å		6.84 Å/pix.
x 128 pix.
= 875.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.48102206 - 1.099214
Average (Standard dev.)0.023298522 (±0.2025067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 875.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.846.846.84
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z875.520875.520875.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.4811.0990.023

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Supplemental data

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Sample components

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Entire : Whole Chlamydomonas cells

EntireName: Whole Chlamydomonas cells
Components
  • Organelle or cellular component: Whole Chlamydomonas cells

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Supramolecule #1: Whole Chlamydomonas cells

SupramoleculeName: 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
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4 / Organelle: Golgi

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: TAP media
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen 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.
DetailsThe cells were frozen onto grids, then thinned using cryo-focused ion beam milling.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: TOM, AV3) / Number subtomograms used: 271
ExtractionNumber tomograms: 29 / Number images used: 271 / Reference model: None / Software: (Name: TOM, AV3)
Details: Subtomograms were extracted in the positions defined by the geometry of the COPI triad positions obtained from determination of the triad structure.
CTF correctionSoftware: (Name: CTFFIND, CTFPHASEFLIP)
Final angle assignmentType: NOT APPLICABLE / Software - Name: AV3

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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