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- EMDB-2523: Structure of the mammalian oligosaccharyl-transferase complex in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2523
TitleStructure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon
Map dataReconstruction was masked
Sample
  • Sample: Canis familiaris Sec61, OST and TRAP bound to a translocating wheat germ 80S ribosome
  • Complex: Triticum aestivum 80S ribosome
  • Protein or peptide: Sec61
  • Protein or peptide: Oligosaccharyltransferase
  • Protein or peptide: Translocon-Associated Protein omplex
KeywordsCo-translational protein translocation / N-Glycosylation
Biological speciesTriticum aestivum (bread wheat) / Canis lupus familiaris (dog)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsPfeffer S / Dudek J / Gogala M / Schorr S / Linxweiler J / Lang S / Becker T / Beckmann R / Zimmermann F
CitationJournal: Nat Commun / Year: 2014
Title: Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon.
Authors: Stefan Pfeffer / Johanna Dudek / Marko Gogala / Stefan Schorr / Johannes Linxweiler / Sven Lang / Thomas Becker / Roland Beckmann / Richard Zimmermann / Friedrich Förster /
Abstract: In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting ...In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.
History
DepositionNov 26, 2013-
Header (metadata) releaseDec 18, 2013-
Map releaseJan 22, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2423.jpg

Downloads & links

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Map

FileDownload / File: emd_2523.map.gz / Format: CCP4 / Size: 804.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction was masked
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.24 Å/pix.
x 600 pix.
= 742.44 Å		1.24 Å/pix.
x 600 pix.
= 742.44 Å		1.24 Å/pix.
x 600 pix.
= 742.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2374 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.41137141 - 0.9818064
Average (Standard dev.)0.00244991 (±0.03538019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-300-300-299
Dimensions600600600
Spacing600600600
CellA=B=C: 742.44006 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23741.23741.2374
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z742.440742.440742.440
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-300-299
NX/NY/NZ600600600
MAP C/R/S213
start NC/NR/NS-300-300-299
NC/NR/NS600600600
D min/max/mean-0.4110.9820.002

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Supplemental data

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Sample components

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Entire : Canis familiaris Sec61, OST and TRAP bound to a translocating whe...

EntireName: Canis familiaris Sec61, OST and TRAP bound to a translocating wheat germ 80S ribosome
Components
  • Sample: Canis familiaris Sec61, OST and TRAP bound to a translocating wheat germ 80S ribosome
  • Complex: Triticum aestivum 80S ribosome
  • Protein or peptide: Sec61
  • Protein or peptide: Oligosaccharyltransferase
  • Protein or peptide: Translocon-Associated Protein omplex

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Supramolecule #1000: Canis familiaris Sec61, OST and TRAP bound to a translocating whe...

SupramoleculeName: Canis familiaris Sec61, OST and TRAP bound to a translocating wheat germ 80S ribosome
type: sample / ID: 1000
Oligomeric state: One 80S ribosome binds one Sec61, one OST and one TRAP
Number unique components: 4

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Supramolecule #1: Triticum aestivum 80S ribosome

SupramoleculeName: Triticum aestivum 80S ribosome / type: complex / ID: 1 / Name.synonym: Wheat germ 80S ribosome / Details: Data-subset resulted from computational sorting / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Triticum aestivum (bread wheat) / synonym: Wheat Germ

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Macromolecule #1: Sec61

MacromoleculeName: Sec61 / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog

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Macromolecule #2: Oligosaccharyltransferase

MacromoleculeName: Oligosaccharyltransferase / type: protein_or_peptide / ID: 2 / Name.synonym: OST / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog

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Macromolecule #3: Translocon-Associated Protein omplex

MacromoleculeName: Translocon-Associated Protein omplex / type: protein_or_peptide / ID: 3 / Name.synonym: TRAP / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog

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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

BufferpH: 7.6
Details: 30 mM Hepes/KOH 7.6, 10 mM Mg(OAc)2, 180 mM KOAC/HAc pH 7.6, 0.3 % Digitonin, 1 mM DTT
GridDetails: Quantifoil grids pre-coated with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJul 17, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 6119 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 148721 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.3 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using SIGNATURE, classified using MAPPOS and processed with SPIDER
CTF correctionDetails: On 3D-volume (SPIDER)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.3 Å / Resolution method: OTHER / Software - Name: SPIDER, Signature, MAPPOS
Details: This datasubset resulted from computational sorting.
Number images used: 15705

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Atomic model buiding 1

Initial modelPDB ID:

2wwb

SoftwareName: Chimera, Coot, MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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