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- EMDB-2432: The structure of the COPII coat assembled on membranes -

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Basic information

Entry
Database: EMDB / ID: EMD-2432
TitleThe structure of the COPII coat assembled on membranes
Map datapos277.mrc
Sample
  • Sample: Cryo-tomogram of COPII-coated membrane
  • Protein or peptide: Sar1p
  • Protein or peptide: Sec23p
  • Protein or peptide: Sec24p
  • Protein or peptide: Sec13p
  • Protein or peptide: Sec31p
KeywordsCOPII / coat / secretion / trafficking / Sec23 / Sec24 / Sar1 / Sec13 / Sec31 / membrane / budding
Function / homology
Function and homology information


COPII vesicle coat / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / intracellular protein transport / ER to Golgi transport vesicle membrane / Golgi membrane / GTPase activity / GTP binding / endoplasmic reticulum membrane / Golgi apparatus ...COPII vesicle coat / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / intracellular protein transport / ER to Golgi transport vesicle membrane / Golgi membrane / GTPase activity / GTP binding / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / zinc ion binding
Similarity search - Function
small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Sec24-like, trunk domain / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain ...small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Sec24-like, trunk domain / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich / Zinc finger, Sec23/Sec24-type superfamily / Sec23/Sec24 helical domain superfamily / Sec23/Sec24 zinc finger / Sec23/Sec24 trunk domain / Sec23/Sec24 helical domain / Sec23/Sec24 beta-sandwich domain / Ras GTPase-activating domain / Gelsolin-like domain superfamily / Roc domain / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Gelsolin-like domain / Gelsolin repeat / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / ARF-like small GTPases; ARF, ADP-ribosylation factor / ADF-H/Gelsolin-like domain superfamily / von Willebrand factor A-like domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Sec24p / Small COPII coat GTPase SAR1 / : / : / :
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM / negative staining
AuthorsZanetti G / Prinz S / Daum S / Meister A / Schekman R / Bacia K / Briggs JAG
CitationJournal: Elife / Year: 2013
Title: The structure of the COPII transport-vesicle coat assembled on membranes.
Authors: Giulia Zanetti / Simone Prinz / Sebastian Daum / Annette Meister / Randy Schekman / Kirsten Bacia / John A G Briggs /
Abstract: Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, ...Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.
History
DepositionJul 29, 2013-
Header (metadata) releaseSep 4, 2013-
Map releaseSep 18, 2013-
UpdateNov 20, 2013-
Current statusNov 20, 2013Processing site: PDBe / Status: Released

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
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Structure viewerEM map:
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Supplemental images

EMD-2432.png

Downloads & links

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Map

FileDownload / File: emd_2432.map.gz / Format: CCP4 / Size: 4.6 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotationpos277.mrc
Voxel sizeX=Y=Z: 4.3 Å
Density
Minimum - Maximum-18760.0 - 14177.0
Average (Standard dev.)222.076065059999991 (±1227.533203129999947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00302
Dimensions20482048600
Spacing20482048600
CellA: 8806.4 Å / B: 8806.4 Å / C: 2580.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z4.34.34.3
M x/y/z20482048600
origin x/y/z0.0000.0000.000
length x/y/z8806.4008806.4002580.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS00302
NC/NR/NS20482048600
D min/max/mean-18760.00014177.000222.076

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Supplemental data

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Sample components

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Entire : Cryo-tomogram of COPII-coated membrane

EntireName: Cryo-tomogram of COPII-coated membrane
Components
  • Sample: Cryo-tomogram of COPII-coated membrane
  • Protein or peptide: Sar1p
  • Protein or peptide: Sec23p
  • Protein or peptide: Sec24p
  • Protein or peptide: Sec13p
  • Protein or peptide: Sec31p

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Supramolecule #1000: Cryo-tomogram of COPII-coated membrane

SupramoleculeName: Cryo-tomogram of COPII-coated membrane / type: sample / ID: 1000 / Number unique components: 5

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Macromolecule #1: Sar1p

MacromoleculeName: Sar1p / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytosol/endoplasmic reticulum
Molecular weightExperimental: 21.437 KDa / Theoretical: 21.437 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: KBB1012 / Recombinant cell: BL21/DE3 / Recombinant plasmid: pTY40
SequenceUniProtKB: Small COPII coat GTPase SAR1 / InterPro: Ras GTPase-activating domain, Roc domain

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Macromolecule #2: Sec23p

MacromoleculeName: Sec23p / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytosol/endoplasmic reticulum
Molecular weightExperimental: 85.437 KDa / Theoretical: 85.437 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RSY3764 / Recombinant plasmid: pTKY9
SequenceUniProtKB: UNIPROTKB: E7QAP0
InterPro: Sec23/Sec24, trunk domain, Sec23/Sec24, helical domain, Sec23/Sec24 beta-sandwich, Zinc finger, Sec23/Sec24-type

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Macromolecule #3: Sec24p

MacromoleculeName: Sec24p / type: protein_or_peptide / ID: 3 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytosol/endoplasmic reticulum
Molecular weightExperimental: 103.577 KDa / Theoretical: 103.577 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RSY3764 / Recombinant plasmid: pLM129
SequenceUniProtKB: Sec24p
InterPro: Sec23/Sec24, trunk domain, Sec23/Sec24 beta-sandwich, Sec23/Sec24, helical domain, Zinc finger, Sec23/Sec24-type

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Macromolecule #4: Sec13p

MacromoleculeName: Sec13p / type: protein_or_peptide / ID: 4 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytosol/endoplasmic reticulum
Molecular weightExperimental: 20.794 KDa / Theoretical: 20.794 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RSY1112 / Recombinant plasmid: pNS3141 (6H31/CK1313)
SequenceUniProtKB: UNIPROTKB: E7Q6Z3

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Macromolecule #5: Sec31p

MacromoleculeName: Sec31p / type: protein_or_peptide / ID: 5 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytosol/endoplasmic reticulum
Molecular weightExperimental: 138.824 KDa / Theoretical: 138.824 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RSY1112 / Recombinant plasmid: pNS3141 (6H31/CK1313)
SequenceUniProtKB: UNIPROTKB: E7Q1I6

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation statefilament

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Sample preparation

BufferpH: 6.8 / Details: HEPES, 50 mM KOAc, 1.2 mM MgCl2
StainingType: NEGATIVE / Details: plunge frozen
GridDetails: C-flat grids
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 19500
Specialist opticsEnergy filter - Name: GATAN GIF 2002
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 3 °
Microscopy ID1
DateSep 18, 2012
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Number real images: 26 / Average electron dose: 80 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 19500
Specialist opticsEnergy filter - Name: GATAN GIF 2002
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 3 °
Microscopy ID2
DateJun 19, 2012
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Number real images: 26 / Average electron dose: 80 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each projection
Final reconstructionSoftware - Name: Imod, Raptor / Number images used: 40
DetailsCTF correction was performed on each projection. Projections aligned based on gold fiducially

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