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- EMDB-62357: Structure of DdmD dimer with ssDNA without nucleotide -

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Basic information

Entry
Database: EMDB / ID: EMD-62357
TitleStructure of DdmD dimer with ssDNA without nucleotide
Map dataDdmd_ssDNA_APO
Sample
  • Complex: DdmD dimer with ssDNA without nucleotide
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • DNA: DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')
KeywordsHelicase/UvrB N-terminal domain-containing protein / DNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA complex
Function / homologyHelicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) (bacteria) / Vibrio cholerae O1 biovar El Tor str. N16961 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsLin ZH / Gao HS / Liu YS / Li RY
Funding support China, 2 items
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, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62357.png

Downloads & links

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Map

FileDownload / File: emd_62357.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDdmd_ssDNA_APO
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 275.789 Å		1.08 Å/pix.
x 256 pix.
= 275.789 Å		1.08 Å/pix.
x 256 pix.
= 275.789 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0773 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-3.4070003 - 6.0137024
Average (Standard dev.)0.0041217515 (±0.15515788)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.7888 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Ddmd ssDNA APO

Fileemd_62357_half_map_1.map
AnnotationDdmd_ssDNA_APO
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Ddmd ssDNA APO

Fileemd_62357_half_map_2.map
AnnotationDdmd_ssDNA_APO
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DdmD dimer with ssDNA without nucleotide

EntireName: DdmD dimer with ssDNA without nucleotide
Components
  • Complex: DdmD dimer with ssDNA without nucleotide
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • DNA: DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')

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Supramolecule #1: DdmD dimer with ssDNA without nucleotide

SupramoleculeName: DdmD dimer with ssDNA without nucleotide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) (bacteria)
Molecular weightTheoretical: 300 KDa

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

MacromoleculeName: Helicase/UvrB N-terminal domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae O1 biovar El Tor str. N16961 (bacteria)
Molecular weightTheoretical: 136.556766 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GPLGSMNVSI EEFTHFDFQL VPEPSPLDLV ITESLKNHIE VNGVKSGALL PLPFQTGIGK TYTALNFLLQ QMLEQVRSEL KEENTGKKS KRLLYYVTDS VDNVVSAKAD LLKLIEKQTV KGEPRFTLEQ QEYLKAQIVH LPNQSEQLLQ CSDAVLNDVL I GFNLNAER ...String:
GPLGSMNVSI EEFTHFDFQL VPEPSPLDLV ITESLKNHIE VNGVKSGALL PLPFQTGIGK TYTALNFLLQ QMLEQVRSEL KEENTGKKS KRLLYYVTDS VDNVVSAKAD LLKLIEKQTV KGEPRFTLEQ QEYLKAQIVH LPNQSEQLLQ CSDAVLNDVL I GFNLNAER DVQAEWSAIS GLRRHASNPE VKISLNRQAG YFYRNLIDRL QKKQKGADRV LLSGSLLASV ETLLPGEKIR NG SAHVAFL TTSKFLKGFH NTRSRYSPLR DLSGAVLIID EIDKQNQVIL SELCKQQAQD LIWAIRTLRA NFRDHQLESS PRY DKIEDL FEPLRERLEE FGTNWNLAFA FNTEGANLNE RPVRLFSDRS FTHVSSATHK LSLKSDFLRR KNLIFSDEKV EGSL IEKHG LLTRFVNEAD VIYQWFLGTM RKAVFQYWEN VRGLEIEVRE NRSLEGTFQE AVQSLLTHFN LQEFESAVYE SFDTR GLRQ SAGGKANKLS SSKSYHHTGL KLVEVAHNQG TRDTVNCKAS FLNTSPSGVL ADMVDAGAVI LGISATARAD TVIHNF DFK YLNERLGNKL LSLSREQKQR VNNYYHSRRN YKDNGVVLTV KYLNSRDAFL DALLEEYKPE ARSSHFILNH YLGIAES EQ AFVRSWLSKL LASIKAFISS PDNRYMLSLL NRTLDTTRQN INDFIQFCCD KWAKEFNVKT KTFFGVNADW MRLVGYDE I SKHLNTELGK VVVFSTYASM GAGKNPDYAV NLALEGESLI SVADVTYSTQ LRSDIDSIYL EKPTQLLLSD DYSHTANQL CQFHQILSLQ ENGELSPKSA ENWCRQQLMG MSRERSLQQY HQTSDYQSAV RKYIEQAVGR AGRTSLKRKQ ILLFVDSGLK EILAEESRD PSLFSHEYVA LVNKAKSAGK SIVEDRAVRR LFNLAQRNNK DGMLSIKALV HRLHNQPASK SDIQEWQDIR T QLLRYPTV AFQPERFNRL YLQSMTKGYY RYQGNLDGDP NSFEFFDRVP YGDMVSEEDC SLATLVQNQY VRPWFERKGF AC SWQKEAN VMTPIMFTNI YKGALGEQAV EAVLTAFDFT FEEVPNSIYE RFDNRVIFAG IEQPIWLDSK YWKHEGNESS EGY SSKIAL VEEEFGPSKF IYVNALGDTS KPIRYLNSCF VETSPQLAKV IEIPALIDDS NADTNRTAVQ ELIKWLHHS

UniProtKB: Helicase/UvrB N-terminal domain-containing protein

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Macromolecule #2: DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*A)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.647446 KDa
SequenceString:
(DA)(DA)(DC)(DA)(DT)(DT)(DA)(DC)(DA)(DA) (DA)(DA)

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Macromolecule #3: DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*AP*CP*AP*TP*TP*AP*CP*AP*AP*AP*AP*T)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.638432 KDa
SequenceString:
(DA)(DC)(DA)(DT)(DT)(DA)(DC)(DA)(DA)(DA) (DA)(DT)

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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

Concentration5 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 5600 / Average electron dose: 50.0 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: 2.0 µm / Nominal defocus min: 1.0 µm
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

Startup modelType of model: OTHER / Details: Ab-initio
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 280039
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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