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- EMDB-41785: DdmD dimer in complex with ssDNA -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-41785
TitleDdmD dimer in complex with ssDNA
Map data
Sample
  • Complex: DdmD
    • Complex: Helicase/UvrB N-terminal domain-containing protein
      • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3')
KeywordsDdmD / helicase / nuclease / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homologyHelicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsBravo JPK / Taylor DW
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_41785.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8332 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.25990602 - 0.5243328
Average (Standard dev.)-0.000030380124 (±0.008374699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 426.5984 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : DdmD

EntireName: DdmD
Components
  • Complex: DdmD
    • Complex: Helicase/UvrB N-terminal domain-containing protein
      • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3')

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Supramolecule #1: DdmD

SupramoleculeName: DdmD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Helicase/UvrB N-terminal domain-containing protein

SupramoleculeName: Helicase/UvrB N-terminal domain-containing protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Vibrio cholerae (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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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 (bacteria)
Molecular weightTheoretical: 136.145297 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNVSIEEFTH FDFQLVPEPS PLDLVITESL KNHIEVNGVK SGALLPLPFQ TGIGKTYTAL NFLLQQMLEQ VRSELKEENT GKKSKRLLY YVTDSVDNVV SAKADLLKLI EKQTVKGEPR FTLEQQEYLK AQIVHLPNQS EQLLQCSDAV LNDVLIGFNL N AERDVQAE ...String:
MNVSIEEFTH FDFQLVPEPS PLDLVITESL KNHIEVNGVK SGALLPLPFQ TGIGKTYTAL NFLLQQMLEQ VRSELKEENT GKKSKRLLY YVTDSVDNVV SAKADLLKLI EKQTVKGEPR FTLEQQEYLK AQIVHLPNQS EQLLQCSDAV LNDVLIGFNL N AERDVQAE WSAISGLRRH ASNPEVKISL NRQAGYFYRN LIDRLQKKQK GADRVLLSGS LLASVETLLP GEKIRNGSAH VA FLTTSKF LKGFHNTRSR YSPLRDLSGA VLIIDEIDKQ NQVILSELCK QQAQDLIWAI RTLRANFRDH QLESSPRYDK IED LFEPLR ERLEEFGTNW NLAFAFNTEG ANLNERPVRL FSDRSFTHVS SATHKLSLKS DFLRRKNLIF SDEKVEGSLI EKHG LLTRF VNEADVIYQW FLGTMRKAVF QYWENVRGLE IEVRENRSLE GTFQEAVQSL LTHFNLQEFE SAVYESFDTR GLRQS AGGK ANKLSSSKSY HHTGLKLVEV AHNQGTRDTV NCKASFLNTS PSGVLADMVD AGAVILGISA TARADTVIHN FDFKYL NER LGNKLLSLSR EQKQRVNNYY HSRRNYKDNG VVLTVKYLNS RDAFLDALLE EYKPEARSSH FILNHYLGIA ESEQAFV RS WLSKLLASIK AFISSPDNRY MLSLLNRTLD TTRQNINDFI QFCCDKWAKE FNVKTKTFFG VNADWMRLVG YDEISKHL N TELGKVVVFS TYASMGAGKN PDYAVNLALE GESLISVADV TYSTQLRSDI DSIYLEKPTQ LLLSDDYSHT ANQLCQFHQ ILSLQENGEL SPKSAENWCR QQLMGMSRER SLQQYHQTSD YQSAVRKYIE QAVGRAGRTS LKRKQILLFV DSGLKEILAE ESRDPSLFS HEYVALVNKA KSAGKSIVED RAVRRLFNLA QRNNKDGMLS IKALVHRLHN QPASKSDIQE WQDIRTQLLR Y PTVAFQPE RFNRLYLQSM TKGYYRYQGN LDGDPNSFEF FDRVPYGDMV SEEDCSLATL VQNQYVRPWF ERKGFACSWQ KE ANVMTPI MFTNIYKGAL GEQAVEAVLT AFDFTFEEVP NSIYERFDNR VIFAGIEQPI WLDSKYWKHE GNESSEGYSS KIA LVEEEF GPSKFIYVNA LGDTSKPIRY LNSCFVETSP QLAKVIEIPA LIDDSNADTN RTAVQELIKW LHHS

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

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Macromolecule #2: DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*AP*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.310177 KDa
SequenceString:
(DT)(DT)(DA)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 299440
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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