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- EMDB-17561: Cryo-ET subtomogram of 70S ribosomes in Escherichia coli cells at... -

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Basic information

Entry
Database: EMDB / ID: EMD-17561
TitleCryo-ET subtomogram of 70S ribosomes in Escherichia coli cells at 37 and 46 degrees centigrade and in Escherichia coli cells overexpressing GroELS and MetK
Map data
Sample
  • Complex: 70S ribosome
KeywordsChaperonin / Folding cage / proteostasis / heat shock / ATPase / CHAPERONE
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 8.4 Å
AuthorsWagner J / Caravajal AI / Beck F / Bracher A / Wan W / Bohn S / Koerner R / Baumeister W / Fernandez-Busnadiego R / Hartl FU
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2024
Title: Visualizing chaperonin function in situ by cryo-electron tomography.
Authors: Jonathan Wagner / Alonso I Carvajal / Andreas Bracher / Florian Beck / William Wan / Stefan Bohn / Roman Körner / Wolfgang Baumeister / Ruben Fernandez-Busnadiego / F Ulrich Hartl /
Abstract: Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor ...Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.
History
DepositionJun 5, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17561.png

Downloads & links

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Map

FileDownload / File: emd_17561.map.gz / Format: CCP4 / Size: 29.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
3.52 Å/pix.
x 198 pix.
= 696.96 Å		3.52 Å/pix.
x 198 pix.
= 696.96 Å		3.52 Å/pix.
x 198 pix.
= 696.96 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.52 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0038
Minimum - Maximum-0.01610911 - 0.031224562
Average (Standard dev.)0.00006790647 (±0.0018056529)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions198198198
Spacing198198198
CellA=B=C: 696.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17561_msk_1.map
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AxesZYX

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Histogram (log scale)

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Half map: #1

Fileemd_17561_half_map_1.map
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Sample components

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Entire : 70S ribosome

EntireName: 70S ribosome
Components
  • Complex: 70S ribosome

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Cryo-ET subtomogram of 70S ribosomes in Escherichia coli cells at 37 and 46 degrees centigrade and in Escherichia coli cells overexpressing GroELS and MetK
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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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
VitrificationCryogen name: ETHANE-PROPANE
DetailsVitrified E. coli Bl21 (DE3) cells

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: STOPGAP / Number subtomograms used: 242080
ExtractionNumber tomograms: 216 / Number images used: 125860
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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