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- EMDB-5524: A pseudoatomic model of the COPII cage obtained from cryo-electro... -

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Basic information

Entry
Database: EMDB / ID: EMD-5524
TitleA pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry
Map dataReconstruction of the Sec13/31 COPII cage
Sample
  • Sample: Oligomeric assembly of Sec13/31
  • Protein or peptide: Sec13R
  • Protein or peptide: Sec31L1
KeywordsSec13 Sec31 COPII
Function / homologyCOPII vesicle coat / intracellular protein transport / WD40 repeat
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsNoble AJ / Zhang Q / O'Donnell J / Hariri H / Bhattacharya N / Marshall AG / Stagg SM
CitationJournal: Nat Struct Mol Biol / Year: 2013
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
DepositionOct 29, 2012-
Header (metadata) releaseNov 21, 2012-
Map releaseMay 22, 2013-
UpdateMay 22, 2013-
Current statusMay 22, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5524_1.png

Downloads & links

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Map

FileDownload / File: emd_5524.map.gz / Format: CCP4 / Size: 89 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the Sec13/31 COPII cage
Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-1.19492173 - 2.95227885
Average (Standard dev.)-0.00631789 (±0.25620237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-144-144-144
Dimensions288288288
Spacing288288288
CellA=B=C: 800.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z800.640800.640800.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-144-144-144
NC/NR/NS288288288
D min/max/mean-1.1952.952-0.006

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Supplemental data

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Sample components

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Entire : Oligomeric assembly of Sec13/31

EntireName: Oligomeric assembly of Sec13/31
Components
  • Sample: Oligomeric assembly of Sec13/31
  • Protein or peptide: Sec13R
  • Protein or peptide: Sec31L1

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Supramolecule #1000: Oligomeric assembly of Sec13/31

SupramoleculeName: 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
Molecular weightTheoretical: 8.08 MDa

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Macromolecule #1: Sec13R

MacromoleculeName: Sec13R / type: protein_or_peptide / ID: 1 / Name.synonym: SEC13 / Number of copies: 2 / Oligomeric state: Heterotetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytosol
Molecular weightTheoretical: 35.4 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFastBac Dual
SequenceGO: intracellular protein transport / InterPro: WD40 repeat

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Macromolecule #2: Sec31L1

MacromoleculeName: Sec31L1 / type: protein_or_peptide / ID: 2 / Name.synonym: SEC31 / Number of copies: 2 / Oligomeric state: Heterotetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytosol
Molecular weightTheoretical: 133 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFastBAC Dual
SequenceGO: COPII vesicle coat / InterPro: WD40 repeat

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-Cl, pH 7.5, 700 mM KOAc, 1 mM MgOAc, 10 mM DTT
GridDetails: Quantifoil R2/1 grids plasma cleaned for 8 s
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 37000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DateJun 24, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 5052 / Average electron dose: 15 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flip of each particle
Final two d classificationNumber classes: 756
Final reconstructionAlgorithm: 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
DetailsThe particles were selected automatically using template matching.

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