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- PDB-8u0u: DdmD dimer in complex with ssDNA -

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Basic information

Entry
Database: PDB / ID: 8u0u
TitleDdmD dimer in complex with ssDNA
Components
  • DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Helicase/UvrB N-terminal domain-containing protein
KeywordsIMMUNE SYSTEM/DNA / DdmD / helicase / nuclease / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homologyDNA / DNA (> 10) / Helicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsBravo, J.P.K.
Funding support United States, 2items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nature / Year: 2024
Title: Plasmid targeting and destruction by the DdmDE bacterial defence system.
Authors: Jack P K Bravo / Delisa A Ramos / Rodrigo Fregoso Ocampo / Caiden Ingram / David W Taylor /
Abstract: Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease ...Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET). Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.
History
DepositionAug 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)278,9114
Polymers278,9114
Non-polymers00
Water00
1


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

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Components

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


Mass: 136145.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: ERS013206_03454 / Production host: Escherichia coli (E. coli) / References: UniProt: B9TSM3
#2: DNA chain DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 3310.177 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1DdmDCOMPLEXall0MULTIPLE SOURCES
2Helicase/UvrB N-terminal domain-containing proteinCOMPLEX#11RECOMBINANT
3DNACOMPLEX#21SYNTHETIC
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 299440 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00718132
ELECTRON MICROSCOPYf_angle_d0.84224560
ELECTRON MICROSCOPYf_dihedral_angle_d42.0962539
ELECTRON MICROSCOPYf_chiral_restr0.052716
ELECTRON MICROSCOPYf_plane_restr0.0043107

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