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- EMDB-5524: A pseudoatomic model of the COPII cage obtained from cryo-electro... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5524 | |||||||||
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Title | A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry | |||||||||
![]() | Reconstruction of the Sec13/31 COPII cage | |||||||||
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![]() | Sec13 Sec31 COPII | |||||||||
Function / homology | COPII vesicle coat / intracellular protein transport / WD40 repeat![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 12.0 Å | |||||||||
![]() | Noble AJ / Zhang Q / O'Donnell J / Hariri H / Bhattacharya N / Marshall AG / Stagg SM | |||||||||
![]() | ![]() Title: A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry. Authors: Alex J Noble / Qian Zhang / Jason O'Donnell / Hanaa Hariri / Nilakshee Bhattacharya / Alan G Marshall / Scott M Stagg / ![]() Abstract: COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and ...COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13-Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13-Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 81.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.6 KB 11.6 KB | Display Display | ![]() |
Images | ![]() | 162.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.8 KB | Display | ![]() |
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Full document | ![]() | 77.9 KB | Display | |
Data in XML | ![]() | 493 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of the Sec13/31 COPII cage | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.78 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Oligomeric assembly of Sec13/31
Entire | Name: Oligomeric assembly of Sec13/31 |
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Components |
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-Supramolecule #1000: Oligomeric assembly of Sec13/31
Supramolecule | Name: Oligomeric assembly of Sec13/31 / type: sample / ID: 1000 Oligomeric state: Each Sec13/31 heterotetramer consists of two Sec13s and two Sec31s. Number unique components: 2 |
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Molecular weight | Theoretical: 8.08 MDa |
-Macromolecule #1: Sec13R
Macromolecule | Name: Sec13R / type: protein_or_peptide / ID: 1 / Name.synonym: SEC13 / Number of copies: 2 / Oligomeric state: Heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 35.4 KDa |
Recombinant expression | Organism: ![]() |
Sequence | GO: intracellular protein transport / InterPro: WD40 repeat |
-Macromolecule #2: Sec31L1
Macromolecule | Name: Sec31L1 / type: protein_or_peptide / ID: 2 / Name.synonym: SEC31 / Number of copies: 2 / Oligomeric state: Heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 133 KDa |
Recombinant expression | Organism: ![]() |
Sequence | GO: COPII vesicle coat / InterPro: WD40 repeat |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 3 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM Tris-Cl, pH 7.5, 700 mM KOAc, 1 mM MgOAc, 10 mM DTT |
Grid | Details: Quantifoil R2/1 grids plasma cleaned for 8 s |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK IV Method: Add 3ul of sample onto Quantifoil R2/1 grids and plasma clean for 8s using a Gatan Solarus plasma cleaner. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Average: 94 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification |
Date | Jun 24, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 5052 / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 37000 |
Sample stage | Specimen holder: liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Details | The particles were selected automatically using template matching. |
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CTF correction | Details: phase flip of each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN, Spider Details: Later refinements used proc3d's automask2 option to mask out densities internal to the Sec13/31 cage. Number images used: 23404 |
Final two d classification | Number classes: 756 |