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- EMDB-3977: Cryo-electron tomogram of the Chlamydomonas reinhardtii Golgi app... -

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Basic information

Entry
Database: EMDB / ID: EMD-3977
TitleCryo-electron tomogram of the Chlamydomonas reinhardtii Golgi apparatus
Map dataCryo-electron tomogram of Chlamydomonas reinhardtii Golgi apparatus
Sample
  • Organelle or cellular component: Whole Chlamydomonas cells
Biological speciesChlamydomonas reinhardtii (plant)
Methodelectron tomography / cryo EM
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
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
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Supplemental images

emd_3977.png

Downloads & links

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Map

FileDownload / File: emd_3977.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-electron tomogram of Chlamydomonas reinhardtii Golgi apparatus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.68 Å/pix.
x 464 pix.
= 6347.52 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 13.68 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3282. - 2183.
Average (Standard dev.)36.845759999999999 (±347.001899999999978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0068
Dimensions928928464
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0406347.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0068
NC/NR/NS928928464
D min/max/mean-3282.0002183.00036.846

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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
Processingelectron tomography
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.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Dose rate: 2 / Focused ion beam - Duration: 2400 sec. / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 5000 / Focused ion beam - Final thickness: 240
Focused ion beam - Details: Starting curent: 0.5 nA, Final current: 0.03 nA. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios DB-FIB. This is not in a list of allowed ...Focused ion beam - Details: Starting curent: 0.5 nA, Final current: 0.03 nA. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios DB-FIB. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

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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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD
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). This is a bin4 (twice binned) tomogram. Three ...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). This is a bin4 (twice binned) tomogram. Three last tilts (56, 58, and 60 degrees) were excluded from the reconstruction.
Number images used: 55
CTF correctionSoftware: (Name: CTFFIND, CTFPHASEFLIP)

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