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- EMDB-18735: CryoEM structure of a GroEL14-GroES7 complex in presence of ADP-B... -

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Basic information

Entry
Database: EMDB / ID: EMD-18735
TitleCryoEM structure of a GroEL14-GroES7 complex in presence of ADP-BeFx with wide GroEL7 trans ring conformation
Map data
Sample
  • Complex: GroEL14-GroES7-MetK
    • Protein or peptide: Chaperonin GroEL
    • Protein or peptide: Co-chaperonin GroES
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: POTASSIUM ION
  • Ligand: water
KeywordsChaperonin / Folding cage / proteostasis / heat shock / ATPase / CHAPERONE
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / 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 / identical protein binding / membrane / metal ion binding / 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 (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 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
DepositionOct 25, 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_18735.png

Downloads & links

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Map

FileDownload / File: emd_18735.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 324 pix.
= 353.16 Å		1.09 Å/pix.
x 324 pix.
= 353.16 Å		1.09 Å/pix.
x 324 pix.
= 353.16 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.373
Minimum - Maximum-1.9794431 - 4.071842
Average (Standard dev.)0.0018486844 (±0.13018622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 353.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18735_msk_1.map
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Half map: #2

Fileemd_18735_half_map_1.map
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Half map: #1

Fileemd_18735_half_map_2.map
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Sample components

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Entire : GroEL14-GroES7-MetK

EntireName: GroEL14-GroES7-MetK
Components
  • Complex: GroEL14-GroES7-MetK
    • Protein or peptide: Chaperonin GroEL
    • Protein or peptide: Co-chaperonin GroES
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: POTASSIUM ION
  • Ligand: water

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Supramolecule #1: GroEL14-GroES7-MetK

SupramoleculeName: GroEL14-GroES7-MetK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: with bound ADP-BeF3 Mg2+ K+
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.03 MDa

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Macromolecule #1: Chaperonin GroEL

MacromoleculeName: Chaperonin GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: chaperonin ATPase
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 57.260504 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK L IAEAMDKV ...String:
AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK L IAEAMDKV GKEGVITVED GTGLQDELDV VEGMQFDRGY LSPYFINKPE TGAVELESPF ILLADKKISN IREMLPVLEA VA KAGKPLL IIAEDVEGEA LATLVVNTMR GIVKVAAVKA PGFGDRRKAM LQDIATLTGG TVISEEIGME LEKATLEDLG QAK RVVINK DTTTIIDGVG EEAAIQGRVA QIRQQIEEAT SDYDREKLQE RVAKLAGGVA VIKVGAATEV EMKEKKARVE DALH ATRAA VEEGVVAGGG VALIRVASKL ADLRGQNEDQ NVGIKVALRA MEAPLRQIVL NCGEEPSVVA NTVKGGDGNY GYNAA TEEY GNMIDMGILD PTKVTRSALQ YAASVAGLMI TTECMVTDLP KNDAADLGAA GGMGGMGGMG GMM

UniProtKB: Chaperonin GroEL

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Macromolecule #2: Co-chaperonin GroES

MacromoleculeName: Co-chaperonin GroES / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 10.400938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MNIRPLHDRV IVKRKEVETK SAGGIVLTGS AAAKSTRGEV LAVGNGRILE NGEVKPLDVK VGDIVIFNDG YGVKSEKIDN EEVLIMSES DILAIVEA

UniProtKB: Co-chaperonin GroES

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 14 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 14 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 5 / Number of copies: 7 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #6: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 6 / Number of copies: 14 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 21 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC7H15NO4SMOPS
200.0 mMKClPotassium chloride
10.0 mMMgCl2Magnesium chloride
5.0 mMC4H10O2S2Dithiothreitol
30.0 mMNaFSodium fluoride
5.0 mMBeSO4Beryllium sulfate
1.0 mMC10H16N5O13P3Adenosine triphosphate
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 5.9 mM n-octyl-beta-D-glucopyranoside were added before vitrification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 34755
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1sx3

source_name: PDB, initial_model_type: experimental model

5opx

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8qxs:
CryoEM structure of a GroEL14-GroES7 complex in presence of ADP-BeFx with wide GroEL7 trans ring conformation

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