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- PDB-8p4r: In situ structure average of GroEL14-GroES14 complexes in Escheri... -

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Basic information

Entry
Database: PDB / ID: 8p4r
TitleIn situ structure average of GroEL14-GroES14 complexes in Escherichia coli cytosol obtained by cryo electron tomography
Components
  • Chaperonin GroEL
  • Co-chaperonin GroES
KeywordsCHAPERONE / Chaperonin / Folding cage / proteostasis / heat shock / ATPase
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / unfolded protein binding / protein-folding chaperone binding / response to heat / protein refolding ...GroEL-GroES complex / chaperonin ATPase / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / unfolded protein binding / protein-folding chaperone binding / response to heat / protein refolding / ATP binding / metal ion binding / cytoplasm
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
ADENOSINE-5'-TRIPHOSPHATE / : / Co-chaperonin GroES / Chaperonin GroEL
Similarity search - Component
Biological speciesEscherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 11.9 Å
AuthorsWagner, J. / Caravajal, A.I. / Beck, F. / Bracher, A. / Wan, W. / Bohn, S. / Koerner, R. / Baumeister, W. / Fernandez-Busnadiego, R. / Hartl, F.U.
Funding support1items
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
DepositionMay 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_DOI / _em_admin.last_update
Revision 1.2Sep 4, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.3Sep 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chaperonin GroEL
B: Chaperonin GroEL
C: Chaperonin GroEL
D: Chaperonin GroEL
E: Chaperonin GroEL
F: Chaperonin GroEL
G: Chaperonin GroEL
O: Co-chaperonin GroES
P: Co-chaperonin GroES
Q: Co-chaperonin GroES
R: Co-chaperonin GroES
S: Co-chaperonin GroES
T: Co-chaperonin GroES
U: Co-chaperonin GroES
H: Chaperonin GroEL
I: Chaperonin GroEL
J: Chaperonin GroEL
K: Chaperonin GroEL
L: Chaperonin GroEL
M: Chaperonin GroEL
N: Chaperonin GroEL
V: Co-chaperonin GroES
W: Co-chaperonin GroES
X: Co-chaperonin GroES
Y: Co-chaperonin GroES
Z: Co-chaperonin GroES
a: Co-chaperonin GroES
b: Co-chaperonin GroES
hetero molecules


Theoretical massNumber of molelcules
Total (without water)955,24870
Polymers947,26028
Non-polymers7,98842
Water7,314406
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 28 molecules ABCDEFGHIJKLMNOPQRSTUVWXYZab

#1: Protein
Chaperonin GroEL


Mass: 57260.504 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Gene: groEL / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140NH65
#2: Protein
Co-chaperonin GroES


Mass: 10400.938 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Gene: groES / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140NEN6

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Non-polymers , 4 types, 448 molecules

#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: K
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 406 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: GroEL14-GroES14 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Vitrified E. coli Bl21 (DE3) cells
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 3 e/Å2 / Avg electron dose per subtomogram: 120 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
11RELIONfinal Euler assignment
13STOPGAP3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 11.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11213 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 216 / Num. of volumes extracted: 125860
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 992.53 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002364428
ELECTRON MICROSCOPYf_angle_d0.50787080
ELECTRON MICROSCOPYf_chiral_restr0.04310640
ELECTRON MICROSCOPYf_plane_restr0.002611284
ELECTRON MICROSCOPYf_dihedral_angle_d4.62419156

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