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- PDB-9ezy: Vibrio cholerae DdmD-DdmE holo complex -

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Basic information

Entry
Database: PDB / ID: 9ezy
TitleVibrio cholerae DdmD-DdmE holo complex
Components
  • 14 nucleotide DNA guide with terminal 5' phosphate
  • Helicase/UvrB N-terminal domain-containing protein
  • Non-target DNA strand
  • Target DNA strand
  • Vibrio cholerae DdmE
KeywordsIMMUNE SYSTEM / Helicase / Nuclease / Complex / Effector / Argonaute
Function / homologyDNA / DNA (> 10) / Helicase/UvrB N-terminal domain-containing protein / Uncharacterized protein
Function and homology information
Biological speciesVibrio cholerae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.56 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. In this study, we used cryogenic electron microscopy to determine the mechanistic ...Seventh-pandemic strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The 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 nontarget DNA strand. In vitro studies indicate 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 1.0Sep 4, 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
B: Vibrio cholerae DdmE
C: 14 nucleotide DNA guide with terminal 5' phosphate
D: Target DNA strand
E: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,1346
Polymers261,1105
Non-polymers241
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

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Protein , 2 types, 2 molecules AB

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


Mass: 136427.594 Da / Num. of mol.: 1
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
#2: Protein Vibrio cholerae DdmE


Mass: 79468.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: VC_1770 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9KR73

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DNA chain , 3 types, 3 molecules CDE

#3: DNA chain 14 nucleotide DNA guide with terminal 5' phosphate


Mass: 4567.998 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The first adenosine nucleotide was added to obtain three oxygen groups on the 5' phosphate of the DNA guide. This nucleotide was not present on the DNA guide used for structure determination.
Source: (synth.) synthetic construct (others)
#4: DNA chain Target DNA strand


Mass: 20341.029 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain Non-target DNA strand


Mass: 20305.029 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 1 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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-DdmE Holocomplex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
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
2225 mMSodium chlorideNaCl1
32.5 mMDesthiobiotinDesthiobiotin1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 60.99 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: 1283702
3D reconstructionResolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229173 / Symmetry type: POINT
Atomic model buildingDetails: Initial model was generated with alpha fold / Source name: AlphaFold / Type: in silico model

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