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- EMDB-5408: The Structure of the Sec13/31 COPII Cage Bound to Sec23 -

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Basic information

Entry
Database: EMDB / ID: EMD-5408
TitleThe Structure of the Sec13/31 COPII Cage Bound to Sec23
Map dataReconstruction of COPII coat
Sample
  • Sample: Sec13/31-23 coat
  • Protein or peptide: COPII
KeywordsCOPII / Sec13/31 / Sec23 / secretory pathway
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 38.0 Å
AuthorsBhattacharya N / O'Donnell J / Stagg SM
CitationJournal: J Mol Biol / Year: 2012
Title: The structure of the Sec13/31 COPII cage bound to Sec23.
Authors: Nilakshee Bhattacharya / Jason O Donnell / Scott M Stagg /
Abstract: Structural studies have revealed some of the organizing principles and mechanisms involved in the assembly of the COPII coat including the location of the Sec23/24 adapter layer. Previous studies, ...Structural studies have revealed some of the organizing principles and mechanisms involved in the assembly of the COPII coat including the location of the Sec23/24 adapter layer. Previous studies, however, were unable to unambiguously determine the positions of Sec23 and Sec24 in the coat. Here, we have determined a cryogenic electron microscopic structure of Sec13/31 together with Sec23. Electron tomography revealed that the binding of Sec23 induces Sec13/31 to form a variety of different geometries including a cuboctahedron, as was previously characterized for Sec13/31 alone. Single-particle reconstruction of the Sec13/31-23 cuboctahedra revealed that the binding of Sec23 induces a conformational change in Sec13/31, resulting in a more extended conformation. Docking Sec23 crystal structures into the electron microscopy map suggested that Sec24 projects its cargo binding surface out into the large open faces of the coat. These results have implications for the mechanisms by which COPII transports large cargos, cargos with large intracellular domains, and for tethering complexes that must project out of the coat in order to interact with their binding partners. Furthermore, Sec23 binds Sec13/31 at two unique sites in the coat, which suggests that each site may have unique roles in the mechanisms of COPII vesiculation.
History
DepositionMar 20, 2012-
Header (metadata) releaseApr 4, 2012-
Map releaseApr 30, 2012-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 4.3
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5408.png

Downloads & links

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Map

FileDownload / File: emd_5408.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of COPII coat
Voxel sizeX=Y=Z: 4.78 Å
Density
Contour LevelBy AUTHOR: 4.3 / Movie #1: 4.3
Minimum - Maximum-7.48428011 - 13.17608929
Average (Standard dev.)0.0 (±0.94375163)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-23-23-23
Dimensions224224224
Spacing224224224
CellA=B=C: 1070.7201 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.784.784.78
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z1070.7201070.7201070.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-23-23-23
NC/NR/NS224224224
D min/max/mean-7.48413.1760.000

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Supplemental data

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Sample components

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Entire : Sec13/31-23 coat

EntireName: Sec13/31-23 coat
Components
  • Sample: Sec13/31-23 coat
  • Protein or peptide: COPII

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Supramolecule #1000: Sec13/31-23 coat

SupramoleculeName: Sec13/31-23 coat / type: sample / ID: 1000 / Oligomeric state: heterotetramer Sec13/31 binds to Sec23 / Number unique components: 3
Molecular weightTheoretical: 7.1 MDa

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Macromolecule #1: COPII

MacromoleculeName: COPII / type: protein_or_peptide / ID: 1 / Details: expressed in insect cells / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / Organelle: cytoplasm
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pfastBAC

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.5
Details: 700 mM potassium acetate, 25 mM HEPES, 1 mM magnesium acetate, 1mM DTT
StainingType: NEGATIVE / Details: Cryo-grids
GridDetails: R 2/2 Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK I / Method: blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: OTHER
DateSep 9, 2010
Image recordingCategory: FILM / Film or detector model: GENERIC CCD / Digitization - Scanner: OTHER / Number real images: 2850 / Average electron dose: 30 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 38.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC,spider,coran / Number images used: 9571

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