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- EMDB-29912: Cryo-EM 3D map of the Mycobacterium tuberculosis Hsp70 protein Dn... -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-29912
TitleCryo-EM 3D map of the Mycobacterium tuberculosis Hsp70 protein DnaK bound to the nucleotide exchange factor GrpE
Map data
Sample
  • Complex: Binary complex of DnaK with nucleotide exchange factor GrpE
    • Protein or peptide: Chaperone protein DnaK
    • Protein or peptide: Protein GrpE
Keywordsheat shock protein 70 / nucleotide exchange factor / protein folding and refolding / CHAPERONE
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)
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_29912.png

Downloads & links

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Map

FileDownload / File: emd_29912.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.011400831 - 2.0328825
Average (Standard dev.)0.00063794554 (±0.018456439)
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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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
    • Protein or peptide: Chaperone protein DnaK
    • Protein or peptide: Protein 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: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: Chaperone protein DnaK

MacromoleculeName: Chaperone protein DnaK / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 66.91068 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MARAVGIDLG TTNSVVSVLE GGDPVVVANS EGSRTTPSIV AFARNGEVLV GQPAKNQAVT NVDRTVRSVK RHMGSDWSIE IDGKKYTAP EISARILMKL KRDAEAYLGE DITDAVITTP AYFNDAQRQA TKDAGQIAGL NVLRIVNEPT AAALAYGLDK G EKEQRILV ...String:
MARAVGIDLG TTNSVVSVLE GGDPVVVANS EGSRTTPSIV AFARNGEVLV GQPAKNQAVT NVDRTVRSVK RHMGSDWSIE IDGKKYTAP EISARILMKL KRDAEAYLGE DITDAVITTP AYFNDAQRQA TKDAGQIAGL NVLRIVNEPT AAALAYGLDK G EKEQRILV FDLGGGTFDV SLLEIGEGVV EVRATSGDNH LGGDDWDQRV VDWLVDKFKG TSGIDLTKDK MAMQRLREAA EK AKIELSS SQSTSINLPY ITVDADKNPL FLDEQLTRAE FQRITQDLLD RTRKPFQSVI ADTGISVSEI DHVVLVGGST RMP AVTDLV KELTGGKEPN KGVNPDEVVA VGAALQAGVL KGEVKDVLLL DVTPLSLGIE TKGGVMTRLI ERNTTIPTKR SETF TTADD NQPSVQIQVY QGEREIAAHN KLLGSFELTG IPPAPRGIPQ IEVTFDIDAN GIVHVTAKDK GTGKENTIRI QEGSG LSKE DIDRMIKDAE AHAEEDRKRR EEADVRNQAE TLVYQTEKFV KEQREAEGGS KVPEDTLNKV DAAVAEAKAA LGGSDI SAI KSAMEKLGQE SQALGQAIYE AAQAASQATG AAHPGGEPGG AHPGSADDVV DAEVVDDGRE AK

UniProtKB: Chaperone protein DnaK

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Macromolecule #2: Protein GrpE

MacromoleculeName: Protein GrpE / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 24.559555 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTDGNQKPDG NSGEQVTVTD KRRIDPETGE VRHVPPGDMP GGTAAADAAH TEDKVAELTA DLQRVQADFA NYRKRALRDQ QAAADRAKA SVVSQLLGVL DDLERARKHG DLESGPLKSV ADKLDSALTG LGLVAFGAEG EDFDPVLHEA VQHEGDGGQG S KPVIGTVM ...String:
MTDGNQKPDG NSGEQVTVTD KRRIDPETGE VRHVPPGDMP GGTAAADAAH TEDKVAELTA DLQRVQADFA NYRKRALRDQ QAAADRAKA SVVSQLLGVL DDLERARKHG DLESGPLKSV ADKLDSALTG LGLVAFGAEG EDFDPVLHEA VQHEGDGGQG S KPVIGTVM RQGYQLGEQV LRHALVGVVD TVVVDAAELE SVDDGTAVAD TAENDQADQG NSADTSGEQA ESEPSGS

UniProtKB: Protein GrpE

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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 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
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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