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- EMDB-2519: Stalled mammalian ribosome bound to canine pancreatic microsomes -

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Basic information

Entry
Database: EMDB / ID: EMD-2519
TitleStalled mammalian ribosome bound to canine pancreatic microsomes
Map dataRibosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
Sample
  • Sample: Ribosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
  • Complex: ER membrane-associated 80S ribosome
  • Protein or peptide: ER protein translocon
Keywordsendoplasmic reticulum / translocon / ribosome / oligosaccharyltransferase / OST / protein-conducting channel / Sec61 / translocon-associated protein complex / TRAP
Biological speciesOryctolagus cuniculus (rabbit) / Canis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 22.0 Å
AuthorsPfeffer S / Dudek J / Gogala M / Schorr S / Linxweiler J / Lang S / Becker T / Beckmann R / Zimmermann R / Foerster 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 25, 2013-
Header (metadata) releaseDec 4, 2013-
Map releaseJan 15, 2014-
UpdateMar 2, 2016-
Current statusMar 2, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.49
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.49
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2519.tif

Downloads & links

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Map

FileDownload / File: emd_2519.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.88 Å/pix.
x 200 pix.
= 576. Å		2.88 Å/pix.
x 200 pix.
= 576. Å		2.88 Å/pix.
x 200 pix.
= 576. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.49 / Movie #1: 1.49
Minimum - Maximum-7.01099348 - 9.998406409999999
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 576.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.882.882.88
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z576.000576.000576.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-7.0119.998-0.000

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Supplemental data

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Sample components

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Entire : Ribosome-stripped canine pancreatic microsomes were populated wit...

EntireName: Ribosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
Components
  • Sample: Ribosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
  • Complex: ER membrane-associated 80S ribosome
  • Protein or peptide: ER protein translocon

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Supramolecule #1000: Ribosome-stripped canine pancreatic microsomes were populated wit...

SupramoleculeName: Ribosome-stripped canine pancreatic microsomes were populated with rabbit ribosomes, stalled on the CMV regulatory peptide.
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: ER membrane-associated 80S ribosome

SupramoleculeName: ER membrane-associated 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte / Location in cell: Cytosol

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Macromolecule #1: ER protein translocon

MacromoleculeName: ER protein translocon / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas / Location in cell: Endoplasmic reticulum

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
GridDetails: Lacey carbon molybdenum grid
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Method: Blot 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateAug 15, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Average electron dose: 60 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 29000
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

DetailsCandidate particles were localized using template matching and classified using constrained principal component analysis focusing on the large ribosomal subunit and the ER membrane. At the selected coordinates, unbinned subtomograms were reconstructed individually from the weighted back-projections and aligned to a template.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: av3, tom, PyTom / Number subtomograms used: 2896
CTF correctionDetails: micrograph

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