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- EMDB-19220: in situ subtomogram average of MEF cell ribosome in the POST state -

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Basic information

Entry
Database: EMDB / ID: EMD-19220
Titlein situ subtomogram average of MEF cell ribosome in the POST state
Map data
Sample
  • Complex: in situ subtomogram average of MEF cell ribosome
Keywordsribosome elongation subtomogram averaging / RIBOSOME
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 20.0 Å
AuthorsFedry J / Forster F
Funding supportEuropean Union, Netherlands, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)724425European Union
Netherlands Organisation for Scientific Research (NWO)724016001 Netherlands
Netherlands Organisation for Scientific Research (NWO)212152 Netherlands
CitationJournal: Mol Cell / Year: 2024
Title: Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells.
Authors: Juliette Fedry / Joana Silva / Mihajlo Vanevic / Stanley Fronik / Yves Mechulam / Emmanuelle Schmitt / Amédée des Georges / William James Faller / Friedrich Förster /
Abstract: Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, ...Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.
History
DepositionDec 21, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19220.png

Downloads & links

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Map

FileDownload / File: emd_19220.map.gz / Format: CCP4 / Size: 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.)
4.34 Å/pix.
x 128 pix.
= 555.52 Å		4.34 Å/pix.
x 128 pix.
= 555.52 Å		4.34 Å/pix.
x 128 pix.
= 555.52 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 4.34 Å
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Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.67710483 - 1.1718566
Average (Standard dev.)0.003318166 (±0.15374018)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 555.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_19220_additional_1.map
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Half map: #1

Fileemd_19220_half_map_1.map
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Half map: #2

Fileemd_19220_half_map_2.map
Projections & Slices
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Sample components

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Entire : in situ subtomogram average of MEF cell ribosome

EntireName: in situ subtomogram average of MEF cell ribosome
Components
  • Complex: in situ subtomogram average of MEF cell ribosome

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Supramolecule #1: in situ subtomogram average of MEF cell ribosome

SupramoleculeName: in situ subtomogram average of MEF cell ribosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: DMEM
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
DetailsMEF cells

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.3 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 63000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.5 µm / Nominal defocus min: 2.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 178
ExtractionNumber tomograms: 96 / Number images used: 28644
Final angle assignmentType: MAXIMUM LIKELIHOOD

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