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- EMDB-41865: DdmDE handover complex -

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Basic information

Entry
Database: EMDB / ID: EMD-41865
TitleDdmDE handover complex
Map data
Sample
  • Complex: DdmD apo dimer
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • DNA: DNA (29-MER)
    • DNA: DNA (5'-D(P*GP*GP*AP*AP*AP*TP*GP*TP*TP*GP*AP*AP*TP*AP*C)-3')
    • Protein or peptide: DdmE
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: MAGNESIUM ION
KeywordsDdmE / DdmD / pAgo / helicase / nuclease / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homologyUncharacterized protein / Helicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 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
DepositionSep 7, 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

Supplemental images

emd_41865.png

Downloads & links

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Map

FileDownload / File: emd_41865.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-0.21728861 - 0.78580683
Average (Standard dev.)0.001000811 (±0.011224429)
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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Additional map: #1

Fileemd_41865_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41865_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41865_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : DdmD apo dimer

EntireName: DdmD apo dimer
Components
  • Complex: DdmD apo dimer
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • DNA: DNA (29-MER)
    • DNA: DNA (5'-D(P*GP*GP*AP*AP*AP*TP*GP*TP*TP*GP*AP*AP*TP*AP*C)-3')
    • Protein or peptide: DdmE
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DdmD apo dimer

SupramoleculeName: DdmD apo dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Vibrio cholerae (bacteria)

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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: 140.011953 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFMYSVFTCR YWRYIMVRLI KHQQLLGEYC MNVSIEEFTH FDFQLVPEPS PLDLVITESL KNHIEVNGVK SGALLPLPFQ TGIGKTYTA LNFLLQQMLE QVRSELKEEN TGKKSKRLLY YVTDSVDNVV SAKADLLKLI EKQTVKGEPR FTLEQQEYLK A QIVHLPNQ ...String:
MFMYSVFTCR YWRYIMVRLI KHQQLLGEYC MNVSIEEFTH FDFQLVPEPS PLDLVITESL KNHIEVNGVK SGALLPLPFQ TGIGKTYTA LNFLLQQMLE QVRSELKEEN TGKKSKRLLY YVTDSVDNVV SAKADLLKLI EKQTVKGEPR FTLEQQEYLK A QIVHLPNQ SEQLLQCSDA VLNDVLIGFN LNAERDVQAE WSAISGLRRH ASNPEVKISL NRQAGYFYRN LIDRLQKKQK GA DRVLLSG SLLASVETLL PGEKIRNGSA HVAFLTTSKF LKGFHNTRSR YSPLRDLSGA VLIIDEIDKQ NQVILSELCK QQA QDLIWA IRTLRANFRD HQLESSPRYD KIEDLFEPLR ERLEEFGTNW NLAFAFNTEG ANLNERPVRL FSDRSFTHVS SATH KLSLK SDFLRRKNLI FSDEKVEGSL IEKHGLLTRF VNEADVIYQW FLGTMRKAVF QYWENVRGLE IEVRENRSLE GTFQE AVQS LLTHFNLQEF ESAVYESFDT RGLRQSAGGK ANKLSSSKSY HHTGLKLVEV AHNQGTRDTV NCKASFLNTS PSGVLA DMV DAGAVILGIS ATARADTVIH NFDFKYLNER LGNKLLSLSR EQKQRVNNYY HSRRNYKDNG VVLTVKYLNS RDAFLDA LL EEYKPEARSS HFILNHYLGI AESEQAFVRS WLSKLLASIK AFISSPDNRY MLSLLNRTLD TTRQNINDFI QFCCDKWA K EFNVKTKTFF GVNADWMRLV GYDEISKHLN TELGKVVVFS TYASMGAGKN PDYAVNLALE GESLISVADV TYSTQLRSD IDSIYLEKPT QLLLSDDYSH TANQLCQFHQ ILSLQENGEL SPKSAENWCR QQLMGMSRER SLQQYHQTSD YQSAVRKYIE QAVGRAGRT SLKRKQILLF VDSGLKEILA EESRDPSLFS HEYVALVNKA KSAGKSIVED RAVRRLFNLA QRNNKDGMLS I KALVHRLH NQPASKSDIQ EWQDIRTQLL RYPTVAFQPE RFNRLYLQSM TKGYYRYQGN LDGDPNSFEF FDRVPYGDMV SE EDCSLAT LVQNQYVRPW FERKGFACSW QKEANVMTPI MFTNIYKGAL GEQAVEAVLT AFDFTFEEVP NSIYERFDNR VIF AGIEQP IWLDSKYWKH EGNESSEGYS SKIALVEEEF GPSKFIYVNA LGDTSKPIRY LNSCFVETSP QLAKVIEIPA LIDD SNADT NRTAVQELIK WLHHS

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

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Macromolecule #4: DdmE

MacromoleculeName: DdmE / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 79.195891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVTPQLEPSS QGPLSTLIEQ ISIDTDWVDR SFAIYCVSYK GIDFSERPKR LVTLASETYK SGSVYCLVKG ANKEACYWVL LPKDSKLDL KDTSLAIKPS SAAELPTWQL ARLLIKAIPK VLSGTMPEIK RFESEGLYYL VKSKKLPKDH SGYELTTVEI D LAPCAALG ...String:
MVTPQLEPSS QGPLSTLIEQ ISIDTDWVDR SFAIYCVSYK GIDFSERPKR LVTLASETYK SGSVYCLVKG ANKEACYWVL LPKDSKLDL KDTSLAIKPS SAAELPTWQL ARLLIKAIPK VLSGTMPEIK RFESEGLYYL VKSKKLPKDH SGYELTTVEI D LAPCAALG FKQTLSMGTK TFSPLSWFTL ENGEVQKKAR FATRYQLDDV GKLVSKSIKG DYIKKPLYSN AKNRIQAIDI TK ESYSGFQ LSKVGILEQF MQDLKQAYGD SVSVKLQRIP GEKHRFVSDT IVKNHYVGLF DALKEHRLVI CDLTENQDTD AAL TLLHGI EHLDINAEIA EVPIRGALNI LIVGNKDTYK SDEEDPYQVY RKKYQDTVFQ SCYPERLWNR QGQPNRHVVE VLLK ELLIK LEVHTRKHLI EYPSGPERCV YYMPQRPKDE SSEVRDEPWP VYASKLVGDE WQYTQATQEE LEDIELDLGN DKRHV FHGF ERSPVIYWPE TGDYAIFIDT GIQMLPEFEA VAERLRELKE GRSQDVPIAL LAQFIEENPE SKVINKLRAI LSEWDD VAP LPFDEFSTIA YKSSDEKQFY DWLREQGFFL KTSIRGQSEG FFNASLGFFY NREQGMYFAG GKGSPQSKIE TFSHLYL IK HSFDALPEEV ENLFDVYHLR HRLPTVTPYP FKHLREYVEM QRFRS

UniProtKB: Uncharacterized protein

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Macromolecule #2: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.777658 KDa
SequenceString:
(DG)(DT)(DA)(DT)(DT)(DC)(DA)(DA)(DC)(DA) (DT)(DT)(DT)(DC)(DC)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: DNA (5'-D(P*GP*GP*AP*AP*AP*TP*GP*TP*TP*GP*AP*AP*TP*AP*C)-3')

MacromoleculeName: DNA (5'-D(P*GP*GP*AP*AP*AP*TP*GP*TP*TP*GP*AP*AP*TP*AP*C)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.657059 KDa
SequenceString:
(DG)(DG)(DA)(DA)(DA)(DT)(DG)(DT)(DT)(DG) (DA)(DA)(DT)(DA)(DC)

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

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.301163 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1497134
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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