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- EMDB-0307: Representative tomogram of the COPII in vitro reconstitution used... -

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Basic information

Entry
Database: EMDB / ID: EMD-0307
TitleRepresentative tomogram of the COPII in vitro reconstitution used for subtomogram averaging
Map data*** This is a dummy file *** Representative unbinned 3D-CTF corrected tomogram used for subtomogram averaging of COPII assembled on membranes.
Sample
  • Complex: In vitro reconstitution of COPII membrane deformation with giant unilamellar vesicles (GUVs).
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsZanetti G / Hutchings J / Hagen WJH
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust109161/Z/15/A United Kingdom
Royal SocietyDH130048 United Kingdom
CitationJournal: Nat Commun / Year: 2018
Title: Subtomogram averaging of COPII assemblies reveals how coat organization dictates membrane shape.
Authors: Joshua Hutchings / Viktoriya Stancheva / Elizabeth A Miller / Giulia Zanetti /
Abstract: Eukaryotic cells employ membrane-bound carriers to transport cargo between compartments in a process essential to cell functionality. Carriers are generated by coat complexes that couple cargo ...Eukaryotic cells employ membrane-bound carriers to transport cargo between compartments in a process essential to cell functionality. Carriers are generated by coat complexes that couple cargo capture to membrane deformation. The COPII coat mediates export from the endoplasmic reticulum by assembling in inner and outer layers, yielding carriers of variable shape and size that allow secretion of thousands of diverse cargo. Despite detailed understanding of COPII subunits, the molecular mechanisms of coat assembly and membrane deformation are unclear. Here we present a 4.9 Å cryo-tomography subtomogram averaging structure of in vitro-reconstituted membrane-bound inner coat. We show that the outer coat (Sec13-Sec31) bridges inner coat subunits (Sar1-Sec23-Sec24), promoting their assembly into a tight lattice. We directly visualize the membrane-embedded Sar1 amphipathic helix, revealing that lattice formation induces parallel helix insertions, yielding tubular curvature. We propose that regulators like the procollagen receptor TANGO1 modulate this mechanism to determine vesicle shape and size.
History
DepositionOct 19, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseNov 7, 2018-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing 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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Supplemental images

emd_0307.png

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Map

FileDownload / File: emd_0307.map.gz / Format: CCP4 / Size: 4.9 KB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation*** This is a dummy file *** Representative unbinned 3D-CTF corrected tomogram used for subtomogram averaging of COPII assembled on membranes.
Voxel size
XYZ
EMDB info.0.000270.000271
CCP4 map header0.000270.000271
EM Navigator Movie #11.3271.3271.327
Density
Minimum - Maximum-32768 - 32767
Average (Standard dev.)-584.7793 (±-1)
SymmetrySpace group: 0
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions370837081666
Spacing370837081666
CellA: 1.0 Å / B: 1.0 Å / C: 1666.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z0.000269687162891050.000269687162891051
M x/y/z370837081666
origin x/y/z0.0000.000-1666.000
length x/y/z1.0001.0001666.000
α/β/γ0.0000.0000.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS370837081666
D min/max/mean-32768.00032767.000-584.779

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Supplemental data

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Sample components

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Entire : In vitro reconstitution of COPII membrane deformation with giant ...

EntireName: In vitro reconstitution of COPII membrane deformation with giant unilamellar vesicles (GUVs).
Components
  • Complex: In vitro reconstitution of COPII membrane deformation with giant unilamellar vesicles (GUVs).

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Supramolecule #1: In vitro reconstitution of COPII membrane deformation with giant ...

SupramoleculeName: In vitro reconstitution of COPII membrane deformation with giant unilamellar vesicles (GUVs).
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statehelical array

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Sample preparation

BufferpH: 6.8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI / Diameter: 5 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.0035 µm / Nominal defocus min: 0.0015 µm / Nominal magnification: 105000
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 reconstructionNumber images used: 40

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