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- EMDB-2515: ER membrane-associated ribosome from HeLa cells after treatment w... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2515 | |||||||||
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Title | ER membrane-associated ribosome from HeLa cells after treatment with TRAP-beta siRNA | |||||||||
![]() | ER membrane-associated ribosome from HeLa cells after treatment with TRAP-beta siRNA | |||||||||
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![]() | endoplasmic reticulum / translocon / ribosome / oligosaccharyltransferase / OST / protein-conducting channel / Sec61 / translocon-associated protein complex / TRAP | |||||||||
Biological species | ![]() | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 40.0 Å | |||||||||
![]() | Pfeffer S / Dudek J / Gogala M / Schorr S / Linxweiler J / Lang S / Becker T / Beckmann R / Zimmermann R / Foerster F | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 28.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 9.6 KB 9.6 KB | Display Display | ![]() |
Images | ![]() | 796 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2514C ![]() 2516C ![]() 2517C ![]() 2518C ![]() 2519C ![]() 2523C C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | ER membrane-associated ribosome from HeLa cells after treatment with TRAP-beta siRNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.88 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : ER membrane-associated ribosome from HeLa cells after treatment w...
Entire | Name: ER membrane-associated ribosome from HeLa cells after treatment with TRAP-beta siRNA |
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Components |
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-Supramolecule #1000: ER membrane-associated ribosome from HeLa cells after treatment w...
Supramolecule | Name: ER membrane-associated ribosome from HeLa cells after treatment with TRAP-beta siRNA type: sample / ID: 1000 / Number unique components: 2 |
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-Supramolecule #1: ER membrane-associated 80S ribosome
Supramolecule | Name: ER membrane-associated 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: ER protein translocon
Macromolecule | Name: ER protein translocon / type: protein_or_peptide / ID: 1 / Recombinant expression: No |
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Source (natural) | Organism: ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | subtomogram averaging |
Aggregation state | particle |
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Sample preparation
Concentration | 2 mg/mL |
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Grid | Details: Lacey carbon molybdenum grid |
Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Method: Blot 3 seconds before plunging. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Date | Sep 5, 2012 |
Image recording | Category: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Average electron dose: 60 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 8.0 µm / Nominal defocus min: 7.0 µm / Nominal magnification: 29000 |
Sample stage | Specimen 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
Details | Candidate 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. |
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Final reconstruction | Applied 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 correction | Details: projection |