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- EMDB-15871: Subtomogram Average of Soluble and ER Membrane-Associated Ribosom... -

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Basic information

Entry
Database: EMDB / ID: EMD-15871
TitleSubtomogram Average of Soluble and ER Membrane-Associated Ribosomes in the Decoding-Sampling State
Map data
Sample
  • Organelle or cellular component: Ribosome in the Decoding-Sampling state
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 4.8 Å
AuthorsGemmer M / Fedry JMM / Forster FG
Funding supportEuropean Union, Netherlands, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)724425European Union
Netherlands Organisation for Scientific Research (NWO)724.016.001 Netherlands
CitationJournal: Nature / Year: 2023
Title: Visualization of translation and protein biogenesis at the ER membrane.
Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C ...Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C Howes / Abhay Kotecha / Juliette Fedry / Friedrich Förster /
Abstract: The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane ...The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.
History
DepositionSep 27, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15871.png

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Map

FileDownload / File: emd_15871.map.gz / Format: CCP4 / Size: 160.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.72 Å/pix.
x 348 pix.
= 599.952 Å		1.72 Å/pix.
x 348 pix.
= 599.952 Å		1.72 Å/pix.
x 348 pix.
= 599.952 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.724 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0017
Minimum - Maximum-0.0038360197 - 0.007679395
Average (Standard dev.)3.8308186e-05 (±0.0004153653)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions348348348
Spacing348348348
CellA=B=C: 599.95197 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15871_half_map_1.map
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AxesZYX

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Half map: #2

Fileemd_15871_half_map_2.map
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Sample components

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Entire : Ribosome in the Decoding-Sampling state

EntireName: Ribosome in the Decoding-Sampling state
Components
  • Organelle or cellular component: Ribosome in the Decoding-Sampling state

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Supramolecule #1: Ribosome in the Decoding-Sampling state

SupramoleculeName: Ribosome in the Decoding-Sampling state / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#16
Details: Soluble and ER membrane-bound ribosomes in the Decoding-Sampling state
Source (natural)Organism: Homo sapiens (human)

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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
BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Details: Manual plunger.
DetailsER-derived vesicles decorated with cytosolic ribosomes.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 79000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 7-7 / Number grids imaged: 8 / Average exposure time: 0.7 sec. / Average electron dose: 2.3 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 869 / Number images used: 134350
Reference model: Subtomogram average of ER membrane-associated ribosome
Method: Template matching / Software - Name: PyTom (ver. 0.994)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.1)
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 26980

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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