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- EMDB-17564: CryoEM structure of a GroEL14-GroES7 cage with encapsulated disor... -

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Basic information

Entry
Database: EMDB / ID: EMD-17564
TitleCryoEM structure of a GroEL14-GroES7 cage with encapsulated disordered substrate MetK in the presence of ADP-BeFx
Map data
Sample
  • Complex: GroEL14-GroES7-MetK
    • Protein or peptide: GroEL
    • Protein or peptide: GroES
    • Protein or peptide: MetK
KeywordsChaperonin / Folding cage / proteostasis / heat shock / ATPase / CHAPERONE
Function / homology
Function and homology information


methionine adenosyltransferase / methionine adenosyltransferase activity / S-adenosylmethionine cycle / S-adenosylmethionine biosynthetic process / GroEL-GroES complex / chaperonin ATPase / virion assembly / potassium ion binding / chaperone cofactor-dependent protein refolding / protein folding chaperone ...methionine adenosyltransferase / methionine adenosyltransferase activity / S-adenosylmethionine cycle / S-adenosylmethionine biosynthetic process / GroEL-GroES complex / chaperonin ATPase / virion assembly / potassium ion binding / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / one-carbon metabolic process / 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 / S-adenosylmethionine synthetase / S-adenosylmethionine synthetase, N-terminal / S-adenosylmethionine synthetase, central domain / S-adenosylmethionine synthetase, C-terminal ...Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / S-adenosylmethionine synthetase / S-adenosylmethionine synthetase, N-terminal / S-adenosylmethionine synthetase, central domain / S-adenosylmethionine synthetase, C-terminal / S-adenosylmethionine synthetase, conserved site / S-adenosylmethionine synthetase superfamily / S-adenosylmethionine synthetase, N-terminal domain / S-adenosylmethionine synthetase, central domain / S-adenosylmethionine synthetase, C-terminal domain / S-adenosylmethionine synthase signature 1. / S-adenosylmethionine synthase signature 2. / 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 / S-adenosylmethionine synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsWagner J / Beck F / Bracher A / Caravajal AI / 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_17564.png

Downloads & links

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Map

FileDownload / File: emd_17564.map.gz / Format: CCP4 / Size: 129.7 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.)
1.07 Å/pix.
x 324 pix.
= 346.68 Å		1.07 Å/pix.
x 324 pix.
= 346.68 Å		1.07 Å/pix.
x 324 pix.
= 346.68 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.405
Minimum - Maximum-1.4115007 - 3.199513
Average (Standard dev.)0.00533586 (±0.1078738)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 346.68002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17564_msk_1.map
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Density Histograms

Histogram

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Half map: #1

Fileemd_17564_half_map_1.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: GroEL
    • Protein or peptide: GroES
    • Protein or peptide: MetK

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Supramolecule #1: GroEL14-GroES7-MetK

SupramoleculeName: GroEL14-GroES7-MetK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / 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: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Recombinant expressionOrganism: 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)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MNIRPLHDRV IVKRKEVETK SAGGIVLTGS AAAKSTRGEV LAVGNGRILE NGEVKPLDVK VGDIVIFND GYGVKSEKID NEEVLIMSES DILAIVEA

UniProtKB: Co-chaperonin GroES

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Macromolecule #3: MetK

MacromoleculeName: MetK / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKHLFTSES VSEGHPDKIA DQISDAVLDA ILEQDPKARV ACETYVKTGM VLVGGEITTS AWVDIEEIT RNTVREIGYV HSDMGFDANS CAVLSAIGKQ SPDINQGVDR ADPLEQGAGD Q GLMFGYAT NETDVLMPAP ITYAHRLVQR QAEVRKNGTL PWLRPDAKSQ ...String:
MAKHLFTSES VSEGHPDKIA DQISDAVLDA ILEQDPKARV ACETYVKTGM VLVGGEITTS AWVDIEEIT RNTVREIGYV HSDMGFDANS CAVLSAIGKQ SPDINQGVDR ADPLEQGAGD Q GLMFGYAT NETDVLMPAP ITYAHRLVQR QAEVRKNGTL PWLRPDAKSQ VTFQYDDGKI VG IDAVVLS TQHSEEIDQK SLQEAVMEEI IKPILPAEWL TSATKFFINP TGRFVIGGPM GDC GLTGRK IIVDTYGGMA RHGGGAFSGK DPSKVDRSAA YAARYVAKNI VAAGLADRCE IQVS YAIGV AEPTSIMVET FGTEKVPSEQ LTLLVREFFD LRPYGLIQML DLLHPIYKET AAYGH FGRE HFPWEKTDKA QLLRDAAGLK

UniProtKB: S-adenosylmethionine synthase

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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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 182106
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

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