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- EMDB-17560: In situ structure average of GroEL7-GroES7 chamber with encapsula... -

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Basic information

Entry
Database: EMDB / ID: EMD-17560
TitleIn situ structure average of GroEL7-GroES7 chamber with encapsulated, ordered substrate in Escherichia coli cytosol obtained by cryo electron tomography
Map data
Sample
  • Complex: GroEL7-GroES7 subcomplex
    • Protein or peptide: GroEL
    • Protein or peptide: GroES
KeywordsChaperonin / Folding cage / proteostasis / heat shock / ATPase / CHAPERONE
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / : / isomerase activity / protein folding chaperone / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding ...GroEL-GroES complex / chaperonin ATPase / virion assembly / : / isomerase activity / protein folding chaperone / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / protein-folding chaperone binding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / membrane / cytosol
Similarity search - Function
Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily ...Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / GroES-like superfamily
Similarity search - Domain/homology
Chaperonin GroEL / Co-chaperonin GroES
Similarity search - Component
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 15.2 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_17560.png

Downloads & links

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Map

FileDownload / File: emd_17560.map.gz / Format: CCP4 / Size: 8 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 128 pix.
= 450.56 Å		3.52 Å/pix.
x 128 pix.
= 450.56 Å		3.52 Å/pix.
x 128 pix.
= 450.56 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: 0.733
Minimum - Maximum-0.7202391 - 1.7795112
Average (Standard dev.)0.0021097253 (±0.16796976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17560_msk_1.map
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AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17560_half_map_1.map
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Sample components

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Entire : GroEL7-GroES7 subcomplex

EntireName: GroEL7-GroES7 subcomplex
Components
  • Complex: GroEL7-GroES7 subcomplex
    • Protein or peptide: GroEL
    • Protein or peptide: GroES

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Supramolecule #1: GroEL7-GroES7 subcomplex

SupramoleculeName: GroEL7-GroES7 subcomplex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: subtomograms of GroEL14-GroES14 and GroEL14-GroES7 complexes in E. coli cytosol were aligned so that GroEL7-GroES7 subcomplexes with density for encapsulated ordered substrate match
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG KLIAEAMDKV ...String:
AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG KLIAEAMDKV GKEGVITVED G TGLQDELD VVEGMQFDRG YLSPYFINKP ETGAVELESP FILLADKKIS NIREMLPVLE AV AKAGKPL LIIAEDVEGE ALATLVVNTM RGIVKVAAVK APGFGDRRKA MLQDIATLTG GTV ISEEIG MELEKATLED LGQAKRVVIN KDTTTIIDGV GEEAAIQGRV AQIRQQIEEA TSDY DREKL QERVAKLAGG VAVIKVGAAT EVEMKEKKAR VEDALHATRA AVEEGVVAGG GVALI RVAS KLADLRGQNE DQNVGIKVAL RAMEAPLRQI VLNCGEEPSV VANTVKGGDG NYGYNA ATE EYGNMIDMGI LDPTKVTRSA LQYAASVAGL MITTECMVTD LPKNDAADLG AAGGMGG MG GMGGMM

UniProtKB: Chaperonin GroEL

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Macromolecule #2: GroES

MacromoleculeName: GroES / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MNIRPLHDRV IVKRKEVETK SAGGIVLTGS AAAKSTRGEV LAVGNGRILE NGEVKPLDVK VGDIVIFND GYGVKSEKID NEEVLIMSES DILAIVEA

UniProtKB: Co-chaperonin GroES

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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: 15.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: STOPGAP / Number subtomograms used: 12255
ExtractionNumber tomograms: 216 / Number images used: 125860
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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