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- PDB-8gb3: Structure of the Mycobacterium tuberculosis Hsp70 protein DnaK bo... -

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Basic information

Entry
Database: PDB / ID: 8gb3
TitleStructure of the Mycobacterium tuberculosis Hsp70 protein DnaK bound to the nucleotide exchange factor GrpE
Components
  • Chaperone protein DnaK
  • Protein GrpE
KeywordsCHAPERONE / heat shock protein 70 / nucleotide exchange factor / protein folding and refolding
Function / homology
Function and homology information


adenyl-nucleotide exchange factor activity / ATP-dependent protein folding chaperone / unfolded protein binding / protein folding / protein-folding chaperone binding / hydrolase activity / protein homodimerization activity / ATP binding / cytoplasm
Similarity search - Function
GrpE nucleotide exchange factor / GrpE nucleotide exchange factor, head / GrpE nucleotide exchange factor, coiled-coil / GrpE / grpE protein signature. / Chaperone DnaK / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site ...GrpE nucleotide exchange factor / GrpE nucleotide exchange factor, head / GrpE nucleotide exchange factor, coiled-coil / GrpE / grpE protein signature. / Chaperone DnaK / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70 family / Hsp70 protein / Heat shock protein 70kD, C-terminal domain superfamily / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Protein GrpE / Chaperone protein DnaK
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsXiao, X. / Li, H.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.content_type
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Chaperone protein DnaK
E: Protein GrpE
F: Protein GrpE


Theoretical massNumber of molelcules
Total (without water)116,0303
Polymers116,0303
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Chaperone protein DnaK / / HSP70 / Heat shock 70 kDa protein / Heat shock protein 70


Mass: 66910.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: dnaK_2, dnaK, E5M05_19850, ERS007703_00955, ERS023446_02581, ERS027651_01905, FCN16_12395, SAMEA2682864_01182, SAMEA2683035_02658
Production host: Escherichia coli (E. coli) / References: UniProt: A0A045JRR0
#2: Protein Protein GrpE / HSP-70 cofactor


Mass: 24559.555 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: grpE, E5M05_19845, E5M52_19255, E5M78_19290, ERS007681_03281, ERS007703_00954, ERS024276_02384, SAMEA2683035_02659
Production host: Escherichia coli (E. coli) / References: UniProt: A0A045J399

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Binary complex of DnaK with nucleotide exchange factor GrpE
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 66 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 15720
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM4image acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9973114
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 240737 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035597
ELECTRON MICROSCOPYf_angle_d0.4857569
ELECTRON MICROSCOPYf_dihedral_angle_d12.3872086
ELECTRON MICROSCOPYf_chiral_restr0.041889
ELECTRON MICROSCOPYf_plane_restr0.0041008

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