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- EMDB-2514: ER membrane-associated ribosome from HeLa cells after treatment w... -

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Basic information

Entry
Database: EMDB / ID: EMD-2514
TitleER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
Map dataER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
Sample
  • Sample: ER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
  • 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 speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 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-
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: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2514.tif

Downloads & links

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Map

FileDownload / File: emd_2514.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
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: 1.0 / Movie #1: 1
Minimum - Maximum-5.90264559 - 9.18686581
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-5.9039.1870.000

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Supplemental data

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Sample components

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Entire : ER membrane-associated ribosome from HeLa cells after treatment w...

EntireName: ER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
Components
  • Sample: ER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
  • Complex: ER membrane-associated 80S ribosome
  • Protein or peptide: ER protein translocon

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Supramolecule #1000: ER membrane-associated ribosome from HeLa cells after treatment w...

SupramoleculeName: ER membrane-associated ribosome from HeLa cells after treatment with non-silencing control siRNA
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: Homo sapiens (human) / Strain: HeLa / synonym: Human / Location in cell: Endoplasmic reticulum

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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: Homo sapiens (human) / Strain: HeLa / synonym: Human / 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
DateOct 31, 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: 8.0 µm / Nominal defocus min: 7.0 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 cross correlation peaks in areas containing rough ER were visually inspected to identify true positive matches. 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: 40.0 Å / Resolution method: OTHER / Software - Name: av3, tom, PyTom / Number subtomograms used: 295
CTF correctionDetails: projection

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