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- EMDB-17422: Density for MetK encapsulated in the GroEL7-GroES7 cage -

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Basic information

Entry
Database: EMDB / ID: EMD-17422
TitleDensity for MetK encapsulated in the GroEL7-GroES7 cage
Map dataDensity for MetK encapsulated into the GroEL7-GroES7 cage
Sample
  • Complex: GroEL14-GroES14-MetK2
    • 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 biosynthetic process / S-adenosylmethionine cycle / potassium ion binding / one-carbon metabolic process / magnesium ion binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
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.
Similarity search - Domain/homology
S-adenosylmethionine synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWagner J / Caravajal AI / Beck F / Bracher A / Wan W / Bohn S / Koerner R / Fernandez-Busnadiego R / Baumeister W / 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
DepositionMay 23, 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_17422.png

Downloads & links

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Map

FileDownload / File: emd_17422.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity for MetK encapsulated into the GroEL7-GroES7 cage
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 196 pix.
= 213.64 Å		1.09 Å/pix.
x 196 pix.
= 213.64 Å		1.09 Å/pix.
x 196 pix.
= 213.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.373
Minimum - Maximum-1.6722784 - 2.8195589
Average (Standard dev.)-0.0028655261 (±0.053255085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 213.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17422_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GroEL14-GroES14-MetK2

EntireName: GroEL14-GroES14-MetK2
Components
  • Complex: GroEL14-GroES14-MetK2
    • Protein or peptide: MetK

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Supramolecule #1: GroEL14-GroES14-MetK2

SupramoleculeName: GroEL14-GroES14-MetK2 / 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: MetK

MacromoleculeName: MetK / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: methionine adenosyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
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 opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: CryoSparc ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 322880
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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