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- PDB-9khv: Structure of DdmD dimer with ssDNA without nucleotide -

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Basic information

Entry
Database: PDB / ID: 9khv
TitleStructure of DdmD dimer with ssDNA without nucleotide
Components
  • DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')
  • DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')
  • Helicase/UvrB N-terminal domain-containing protein
KeywordsDNA BINDING PROTEIN/DNA / Helicase/UvrB N-terminal domain-containing protein / DNA BINDING PROTEIN-DNA complex
Function / homologyDNA / DNA (> 10) / Helicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae O1 biovar El Tor str. N16961 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsLin, Z.H. / Gao, H.S. / Liu, Y.S. / Li, R.Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971222 China
National Natural Science Foundation of China (NSFC)32271258 China
CitationJournal: Nucleic Acids Res / Year: 2025
Title: A gate-clamp mechanism for ssDNA translocation by DdmD in Vibrio cholerae plasmid defense.
Authors: Ruoyu Li / Yusong Liu / Haishan Gao / Zhonghui Lin /
Abstract: The DdmDE antiplasmid system, consisting of the helicase-nuclease DdmD and the prokaryotic Argonaute (pAgo) protein DdmE, plays a crucial role in defending Vibrio cholerae against plasmids. Guided by ...The DdmDE antiplasmid system, consisting of the helicase-nuclease DdmD and the prokaryotic Argonaute (pAgo) protein DdmE, plays a crucial role in defending Vibrio cholerae against plasmids. Guided by DNA, DdmE specifically targets plasmids, disassembles the DdmD dimer, and forms a DdmD-DdmE handover complex to facilitate plasmid degradation. However, the precise ATP-dependent DNA translocation mechanism of DdmD has remained unclear. Here, we present cryo-EM structures of DdmD bound to single-stranded DNA (ssDNA) in nucleotide-free, ATPγS-bound, and ADP-bound states. These structures, combined with biochemical analysis, reveal a unique "gate-clamp" mechanism for ssDNA translocation by DdmD. Upon ATP binding, arginine finger residues R855 and R858 reorient to interact with the γ-phosphate, triggering HD2 domain movement. This shift repositions the gate residue Q781, causing a flip of the 3' flank base, which is then clamped by residue F639. After ATP hydrolysis, the arginine finger releases the nucleotide, inducing HD2 to return to its open state. This conformational change enables DdmD to translocate along ssDNA by one nucleotide in the 5' to 3' direction. This study provides new insights into the ATP-dependent translocation of DdmD and contributes to understanding the mechanistic diversity within SF2 helicases.
History
DepositionNov 11, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Helicase/UvrB N-terminal domain-containing protein
C: DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')
B: Helicase/UvrB N-terminal domain-containing protein
D: DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')


Theoretical massNumber of molelcules
Total (without water)280,3994
Polymers280,3994
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Helicase/UvrB N-terminal domain-containing protein


Mass: 136556.766 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O1 biovar El Tor str. N16961 (bacteria)
Gene: VC_1771 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q9KR72
#2: DNA chain DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')


Mass: 3647.446 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')


Mass: 3638.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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: DdmD dimer with ssDNA without nucleotide / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding 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: 2000 nm / Nominal defocus min: 1000 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 recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5600

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487:model refinement
3EPUimage acquisition
5cryoSPARCCTF correction
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 280039 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319883
ELECTRON MICROSCOPYf_angle_d0.50826978
ELECTRON MICROSCOPYf_dihedral_angle_d12.2152820
ELECTRON MICROSCOPYf_chiral_restr0.042972
ELECTRON MICROSCOPYf_plane_restr0.0043405

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