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- PDB-9i91: Ku from Mycobacterium tuberculosis bound to DNA -

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Basic information

Entry
Database: PDB / ID: 9i91
TitleKu from Mycobacterium tuberculosis bound to DNA
Components
  • DNA 1
  • DNA 2
  • Non-homologous end joining protein Ku
KeywordsDNA BINDING PROTEIN / DNA repair / tuberculosis / NHEJ
Function / homology
Function and homology information


positive regulation of ligase activity / DNA helicase complex / double-strand break repair via nonhomologous end joining / double-stranded DNA binding / DNA recombination / protein homodimerization activity
Similarity search - Function
Non-homologous end joining protein Ku, prokaryotic type / Ku70/Ku80 beta-barrel domain / Ku70 and Ku80 are 70kDa and 80kDa subunits of the Lupus Ku autoantigen / Ku70/Ku80 beta-barrel domain / SPOC-like, C-terminal domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Non-homologous end joining protein Ku
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsChaplin, A.K. / Zahid, S.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/X00029X/1 United Kingdom
The Lister Institute of Preventive Medicine United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Oligomerisation of Ku from Mycobacterium tuberculosis promotes DNA synapsis.
Authors: Sayma Zahid / Sonia Baconnais / Henrietta Smith / Saseela Atwal / Lucy Bates / Harriet Read / Ankita Chadda / Florian Morati / Tom Bedwell / Emil G P Stender / Joanne Walter / Steven W ...Authors: Sayma Zahid / Sonia Baconnais / Henrietta Smith / Saseela Atwal / Lucy Bates / Harriet Read / Ankita Chadda / Florian Morati / Tom Bedwell / Emil G P Stender / Joanne Walter / Steven W Hardwick / Fredrik Westerlund / Eric Galburt / Eric Le Cam / Alice Pyne / Galina V Mukamolova / Amanda K Chaplin /
Abstract: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is estimated to infect nearly one-quarter of the global population. A key factor in its resilience and persistence is its ...Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is estimated to infect nearly one-quarter of the global population. A key factor in its resilience and persistence is its robust DNA repair capacity. Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in many organisms, including Mtb, where it is mediated by the Ku protein and the multifunctional LigD enzyme. In this study, we demonstrate that Ku is essential for mycobacterial survival under DNA-damaging conditions. Using cryogenic electron microscopy (cryo-EM), we solved high-resolution structures of both the apo and DNA-bound forms of the Ku-Mtb homodimer. Our structural and biophysical analyses reveal that Ku forms an extended proteo-filament upon binding DNA. We identify critical residues involved in filament formation and DNA synapsis and show that their mutation severely impairs bacterial viability. Furthermore, we propose a model in which the C-terminus of Ku regulates DNA binding and loading and facilitates subsequent recruitment of LigD. These findings provide unique insights into bacterial DNA repair and guide future therapeutics.
History
DepositionFeb 6, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Non-homologous end joining protein Ku
B: Non-homologous end joining protein Ku
C: Non-homologous end joining protein Ku
D: Non-homologous end joining protein Ku
E: Non-homologous end joining protein Ku
F: Non-homologous end joining protein Ku
G: DNA 1
H: DNA 2
I: DNA 1
J: DNA 2


Theoretical massNumber of molelcules
Total (without water)240,23710
Polymers240,23710
Non-polymers00
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Non-homologous end joining protein Ku / Mt-Ku


Mass: 33477.570 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: mku, Rv0937c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WKD9
#2: DNA chain DNA 1


Mass: 9947.389 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: DNA chain DNA 2


Mass: 9738.257 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: KuTB-DNA complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris-HCl1
275 mMSodium ChlorideNaCl1
35 mMMagnesium ChlorideMgCl21
42 mMDithiothreitolDTT1
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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147904 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 161.18 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412431
ELECTRON MICROSCOPYf_angle_d0.670617317
ELECTRON MICROSCOPYf_chiral_restr0.04262007
ELECTRON MICROSCOPYf_plane_restr0.00471958
ELECTRON MICROSCOPYf_dihedral_angle_d20.33864480

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