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- EMDB-41781: DdmE in complex with guide and target DNA -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-41781
TitleDdmE in complex with guide and target DNA
Map data
Sample
  • Complex: DdmE
    • Protein or peptide: DdmE
    • DNA: DNA (5'-D(P*GP*TP*TP*AP*GP*AP*CP*TP*TP*TP*AP*AP*GP*T)-3')
    • DNA: DNA (5'-D(P*AP*CP*TP*TP*AP*AP*AP*GP*TP*CP*TP*AP*AP*CP*CP*TP*AP*TP*AP*GP*GP*AP*T)-3')
    • DNA: DNA (5'-D(P*AP*TP*CP*CP*TP*AP*TP*AP*GP*GP*A)-3')
  • Ligand: MAGNESIUM ION
KeywordsDdmE / Argonaute / pAgo / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homologyUncharacterized protein
Function and homology information
Biological speciesStreptococcus pyogenes (bacteria) / Vibrio cholerae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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 3, 2024-
Map releaseJul 3, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41781.png

Downloads & links

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Map

FileDownload / File: emd_41781.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.49
Minimum - Maximum-3.6440191 - 4.627801
Average (Standard dev.)-0.000000000000338 (±0.061327696)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41781_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41781_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DdmE

EntireName: DdmE
Components
  • Complex: DdmE
    • Protein or peptide: DdmE
    • DNA: DNA (5'-D(P*GP*TP*TP*AP*GP*AP*CP*TP*TP*TP*AP*AP*GP*T)-3')
    • DNA: DNA (5'-D(P*AP*CP*TP*TP*AP*AP*AP*GP*TP*CP*TP*AP*AP*CP*CP*TP*AP*TP*AP*GP*GP*AP*T)-3')
    • DNA: DNA (5'-D(P*AP*TP*CP*CP*TP*AP*TP*AP*GP*GP*A)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DdmE

SupramoleculeName: DdmE / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 211.54 KDa

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Macromolecule #1: DdmE

MacromoleculeName: DdmE / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 78.125672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QGPLSTLIEQ ISIDTDWVDR SFAIYCVSYK GIDFSERPKR LVTLASETYK SGSVYCLVKG ANKEACYWVL LPKDSKLDLK DTSLAIKPS SAAELPTWQL ARLLIKAIPK VLSGTMPEIK RFESEGLYYL VKSKKLPKDH SGYELTTVEI DLAPCAALGF K QTLSMGTK ...String:
QGPLSTLIEQ ISIDTDWVDR SFAIYCVSYK GIDFSERPKR LVTLASETYK SGSVYCLVKG ANKEACYWVL LPKDSKLDLK DTSLAIKPS SAAELPTWQL ARLLIKAIPK VLSGTMPEIK RFESEGLYYL VKSKKLPKDH SGYELTTVEI DLAPCAALGF K QTLSMGTK TFSPLSWFTL ENGEVQKKAR FATRYQLDDV GKLVSKSIKG DYIKKPLYSN AKNRIQAIDI TKESYSGFQL SK VGILEQF MQDLKQAYGD SVSVKLQRIP GEKHRFVSDT IVKNHYVGLF DALKEHRLVI CDLTENQDTD AALTLLHGIE HLD INAEIA EVPIRGALNI LIVGNKDTYK SDEEDPYQVY RKKYQDTVFQ SCYPERLWNR QGQPNRHVVE VLLKELLIKL EVHT RKHLI EYPSGPERCV YYMPQRPKDE SSEVRDEPWP VYASKLVGDE WQYTQATQEE LEDIELDLGN DKRHVFHGFE RSPVI YWPE TGDYAIFIDT GIQMLPEFEA VAERLRELKE GRSQDVPIAL LAQFIEENPE SKVINKLRAI LSEWDDVAPL PFDEFS TIA YKSSDEKQFY DWLREQGFFL KTSIRGQSEG FFNASLGFFY NREQGMYFAG GKGSPQSKIE TFSHLYLIKH SFDALPE EV ENLFDVYHLR HRLPTVTPYP FKHLREYVEM QRFRS

UniProtKB: Uncharacterized protein

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

MacromoleculeName: DNA (5'-D(P*GP*TP*TP*AP*GP*AP*CP*TP*TP*TP*AP*AP*GP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.309826 KDa
SequenceString:
(DG)(DT)(DT)(DA)(DG)(DA)(DC)(DT)(DT)(DT) (DA)(DA)(DG)(DT)

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Macromolecule #3: DNA (5'-D(P*AP*CP*TP*TP*AP*AP*AP*GP*TP*CP*TP*AP*AP*CP*CP*TP*AP*TP...

MacromoleculeName: DNA (5'-D(P*AP*CP*TP*TP*AP*AP*AP*GP*TP*CP*TP*AP*AP*CP*CP*TP*AP*TP*AP*GP*GP*AP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.047598 KDa
SequenceString:
(DA)(DC)(DT)(DT)(DA)(DA)(DA)(DG)(DT)(DC) (DT)(DA)(DA)(DC)(DC)(DT)(DA)(DT)(DA)(DG) (DG)(DA)(DT)

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Macromolecule #4: DNA (5'-D(P*AP*TP*CP*CP*TP*AP*TP*AP*GP*GP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*TP*CP*CP*TP*AP*TP*AP*GP*GP*A)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.357223 KDa
SequenceString:
(DA)(DT)(DC)(DC)(DT)(DA)(DT)(DA)(DG)(DG) (DA)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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

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: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 594426
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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