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- EMDB-29913: Cryo-EM 3D map of the Mycobacterium tuberculosis Hsp70 protein Dn... -

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Basic information

Entry
Database: EMDB / ID: EMD-29913
TitleCryo-EM 3D map of the Mycobacterium tuberculosis Hsp70 protein DnaK bound to the nucleotide exchange factor GrpE
Map dataprocessed by DeepEMhancer
Sample
  • Complex: Binary complex of DnaK with nucleotide exchange factor GrpE
Keywordsheat shock protein 70 / nucleotide exchange factor / protein folding and refolding / CHAPERONE
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsXiao X / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structure of the M. tuberculosis DnaK-GrpE complex reveals how key DnaK roles are controlled.
Authors: Xiansha Xiao / Allison Fay / Pablo Santos Molina / Amanda Kovach / Michael S Glickman / Huilin Li /
Abstract: The molecular chaperone DnaK is essential for viability of Mycobacterium tuberculosis (Mtb). DnaK hydrolyzes ATP to fold substrates, and the resulting ADP is exchanged for ATP by the nucleotide ...The molecular chaperone DnaK is essential for viability of Mycobacterium tuberculosis (Mtb). DnaK hydrolyzes ATP to fold substrates, and the resulting ADP is exchanged for ATP by the nucleotide exchange factor GrpE. It has been unclear how GrpE couples DnaK's nucleotide exchange with substrate release. Here we report a cryo-EM analysis of GrpE bound to an intact Mtb DnaK, revealing an asymmetric 1:2 DnaK-GrpE complex. The GrpE dimer ratchets to modulate both DnaK nucleotide-binding domain and the substrate-binding domain. We further show that the disordered GrpE N-terminus is critical for substrate release, and that the DnaK-GrpE interface is essential for protein folding activity both in vitro and in vivo. Therefore, the Mtb GrpE dimer allosterically regulates DnaK to concomitantly release ADP in the nucleotide-binding domain and substrate peptide in the substrate-binding domain.
History
DepositionFeb 24, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29913.png

Downloads & links

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Map

FileDownload / File: emd_29913.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprocessed by DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.0017791484 - 2.0322707
Average (Standard dev.)0.0006410899 (±0.01834768)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29913_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29913_half_map_2.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 : Binary complex of DnaK with nucleotide exchange factor GrpE

EntireName: Binary complex of DnaK with nucleotide exchange factor GrpE
Components
  • Complex: Binary complex of DnaK with nucleotide exchange factor GrpE

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Supramolecule #1: Binary complex of DnaK with nucleotide exchange factor GrpE

SupramoleculeName: Binary complex of DnaK with nucleotide exchange factor GrpE
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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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

BufferpH: 8
GridModel: Quantifoil R2/1 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 15720 / Average exposure time: 1.5 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 9973114
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4ani

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 240737
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC

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