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- EMDB-2428: The structure of the COPII coat assembled on membranes -

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Basic information

Entry
Database: EMDB / ID: EMD-2428
TitleThe structure of the COPII coat assembled on membranes
Map dataReconstruction of the COPII inner coat assembled on tubular membranes
Sample
  • Sample: Sec23/24-Sar1 complex on membrane
  • Protein or peptide: Sar1p
  • Protein or peptide: Sec23p
  • Protein or peptide: Sec24p
KeywordsCOPII / coat / secretion / trafficking / Sec23 / Sec24 / Sar1 / membrane
Function / homology
Function and homology information


Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Cargo concentration in the ER / regulation of COPII vesicle coating / positive regulation of ER to Golgi vesicle-mediated transport / mitochondria-associated endoplasmic reticulum membrane contact site / nuclear envelope organization / COPII-mediated vesicle transport / vesicle organization / COPII-coated vesicle cargo loading / COPII vesicle coat ...Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Cargo concentration in the ER / regulation of COPII vesicle coating / positive regulation of ER to Golgi vesicle-mediated transport / mitochondria-associated endoplasmic reticulum membrane contact site / nuclear envelope organization / COPII-mediated vesicle transport / vesicle organization / COPII-coated vesicle cargo loading / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / membrane organization / signal sequence binding / mitochondrial fission / fungal-type vacuole membrane / mitochondrial membrane organization / reticulophagy / endoplasmic reticulum exit site / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / GTPase activator activity / SNARE binding / macroautophagy / intracellular protein transport / ER to Golgi transport vesicle membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Golgi membrane / GTPase activity / endoplasmic reticulum membrane / GTP binding / Golgi apparatus / endoplasmic reticulum / mitochondrion / zinc ion binding
Similarity search - Function
small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Protein transport protein Sec23 / Sec23, C-terminal / : / Sec24-like, trunk domain / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich ...small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Protein transport protein Sec23 / Sec23, C-terminal / : / 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 / small GTPase Arf family profile. / Roc domain / Gelsolin-like domain / Gelsolin repeat / Sar1p-like members of the Ras-family of small GTPases / 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 / : / Protein transport protein SEC23 / Small COPII coat GTPase SAR1 / Protein transport protein SEC24
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 23.0 Å
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 26, 2013-
Header (metadata) releaseAug 7, 2013-
Map releaseSep 18, 2013-
UpdateOct 9, 2013-
Current statusOct 9, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bzi
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4bzi
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2428.png

Downloads & links

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Map

FileDownload / File: emd_2428.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the COPII inner coat assembled on tubular membranes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.3 Å/pix.
x 64 pix.
= 275.2 Å		4.3 Å/pix.
x 64 pix.
= 275.2 Å		4.3 Å/pix.
x 64 pix.
= 275.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.4 / Movie #1: 1.4
Minimum - Maximum-2.50167155 - 3.68605518
Average (Standard dev.)0.0 (±0.99999809)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.34.34.3
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z275.200275.200275.200
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-2.5023.6860.000

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Supplemental data

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Sample components

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Entire : Sec23/24-Sar1 complex on membrane

EntireName: Sec23/24-Sar1 complex on membrane
Components
  • Sample: Sec23/24-Sar1 complex on membrane
  • Protein or peptide: Sar1p
  • Protein or peptide: Sec23p
  • Protein or peptide: Sec24p

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Supramolecule #1000: Sec23/24-Sar1 complex on membrane

SupramoleculeName: Sec23/24-Sar1 complex on membrane / type: sample / ID: 1000 / Oligomeric state: array of Sec23/24-Sar1 heterotrimers / Number unique components: 4
Molecular weightExperimental: 210 KDa / Theoretical: 210 KDa

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Macromolecule #1: Sar1p

MacromoleculeName: Sar1p / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: in array / 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 / Number of copies: 3 / Oligomeric state: in array / 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 / Number of copies: 3 / Oligomeric state: in array / 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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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation statehelical array

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Sample preparation

Concentration0.03 mg/mL
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

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GATAN GIF 2002
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 19500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GATAN GIF 2002
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 19500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailssee materials and methods in relevant publication
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: TOM/AV3, Matlab
Details: final map was average of two datasets independently processed
Number subtomograms used: 5000
CTF correctionDetails: each tilted image within tomogram
Final angle assignmentDetails: 0 0 0 in zyz convention

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Atomic model buiding 1

Initial modelPDB ID:

1m2o

SoftwareName: Chimera
Detailsthe map was combined with 1M2V
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross- correlation
Output model

PDB-4bzi:
The structure of the COPII coat assembled on membranes

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Atomic model buiding 2

Initial modelPDB ID:

1m2v

SoftwareName: Chimera
Detailsthe map was combined with 1M2O
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross- correlation
Output model

PDB-4bzi:
The structure of the COPII coat assembled on membranes

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