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- PDB-9ezx: Vibrio cholerae DdmD apo complex -

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Basic information

Entry
Database: PDB / ID: 9ezx
TitleVibrio cholerae DdmD apo complex
ComponentsHelicase/UvrB N-terminal domain-containing protein
KeywordsIMMUNE SYSTEM / Helicase / Nuclease / Complex / Effector
Function / homologyHelicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsLoeff, L. / Jinek, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)820150European Union
CitationJournal: Science / Year: 2024
Title: Molecular mechanism of plasmid elimination by the DdmDE defense system.
Authors: Luuk Loeff / David W Adams / Christelle Chanez / Sandrine Stutzmann / Laurie Righi / Melanie Blokesch / Martin Jinek /
Abstract: Seventh pandemic strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryogenic electron microscopy to reveal the mechanistic basis for plasmid ...Seventh pandemic strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryogenic electron microscopy to reveal the mechanistic basis for plasmid defense by DdmDE. A structure of the DdmD helicase-nuclease reveals it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. In vitro studies reveal that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.
History
DepositionApr 14, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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


Theoretical massNumber of molelcules
Total (without water)272,8552
Polymers272,8552
Non-polymers00
Water00
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 Helicase/UvrB N-terminal domain-containing protein


Mass: 136427.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: VC_1771 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9KR72

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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: Dimeric complex of the DdmD protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.266 MDa / Experimental value: YES
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris hydrochlorideTris-HCl1
2350 mMSodium chlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 / Calibrated magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.98 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1.particle selection
2EPUimage acquisition
4cryoSPARC4.4.1.CTF correction
7Coot0.9model fitting
9PHENIXmodel refinement
11cryoSPARC4.4.1.final Euler assignment
12cryoSPARC4.4.1.classification
13cryoSPARC4.4.1.3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2023352
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184227 / Symmetry type: POINT
Atomic model buildingDetails: Initial model was generated with alpha fold / Source name: AlphaFold / Type: in silico model

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