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- EMDB-50090: Vibrio cholerae DdmD apo complex -

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Basic information

Entry
Database: EMDB / ID: EMD-50090
TitleVibrio cholerae DdmD apo complex
Map dataUnsharpened map
Sample
  • Complex: Dimeric complex of the DdmD protein
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
KeywordsHelicase / Nuclease / Complex / Effector / IMMUNE SYSTEM
Function / homologyHelicase/UvrB N-terminal domain-containing protein
Function and homology information
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsLoeff L / Jinek M
Funding supportEuropean Union, 1 items
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, 2024-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50090.png

Downloads & links

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Map

FileDownload / File: emd_50090.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 576 pix.
= 374.4 Å		0.65 Å/pix.
x 576 pix.
= 374.4 Å		0.65 Å/pix.
x 576 pix.
= 374.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0299
Minimum - Maximum-0.077990845 - 0.2035368
Average (Standard dev.)0.00002018019 (±0.0033595234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50090_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened EM map

Fileemd_50090_additional_1.map
AnnotationSharpened EM map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_50090_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_50090_half_map_2.map
AnnotationHalf map A
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 : Dimeric complex of the DdmD protein

EntireName: Dimeric complex of the DdmD protein
Components
  • Complex: Dimeric complex of the DdmD protein
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein

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Supramolecule #1: Dimeric complex of the DdmD protein

SupramoleculeName: Dimeric complex of the DdmD protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 266 KDa

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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: 136.427594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMNVSIEE FTHFDFQLVP EPSPLDLVIT EPLKNHIEVN GVKSGALLPL PFQTGIGKTY TALNFLLQQM LEQVRSELKE ENTGKKSKR LLYYVTDSVD NVVSAKADLL KLIEKQTVKG EPRFTLEQQE YLKAQIVHLP NQSEQLLQCS DAVLNDVLIG F NLNAERDV ...String:
SNAMNVSIEE FTHFDFQLVP EPSPLDLVIT EPLKNHIEVN GVKSGALLPL PFQTGIGKTY TALNFLLQQM LEQVRSELKE ENTGKKSKR LLYYVTDSVD NVVSAKADLL KLIEKQTVKG EPRFTLEQQE YLKAQIVHLP NQSEQLLQCS DAVLNDVLIG F NLNAERDV QAEWSAISGL RRHASNPEVK ISLNRQAGYF YRNLIDRLQK KQKGADRVLL SGSLLASVET LLPGEKIRNG SA HVAFLTT SKFLKGFHNT RSRYSPLRDL SGAVLIIDEI DKQNQVILSE LCKQQAQDLI WAIRTLRANF RDHQLESSPR YDK IEDLFE PLRERLEEFG TNWNLAFAFN TEGANLNERP VRLFSDRSFT HVSSATHKLS LKSDFLRRKN LIFSDEKVEG SLIE KHGLL TRFVNEADVI YQWFLGTMRK AVFQYWENVR GLEIEVRENR SLEGTFQEAV QSLLTHFNLQ EFESAVYESF DTRGL RQSA GGKANKLSSS KSYHHTGLKL VEVAHNQGTR DTVNCKASFL NTSPSGVLAD MVDAGAVILG ISATARADTV IHNFDF KYL NERLGNKLLS LSREQKQRVN NYYHSRRNYK DNGVVLTVKY LNSRDAFLDA LLEEYKPEAR SSHFILNHYL GIAESEQ AF VRSWLSKLLA SIKAFISSPD NRYMLSLLNR TLDTTRQNIN DFIQFCCDKW AKEFNVKTKT FFGVNADWMR LVGYDEIS K HLNTELGKVV VFSTYASMGA GKNPDYAVNL ALEGESLISV ADVTYSTQLR SDIDSIYLEK PTQLLLSDDY SHTANQLCQ FHQILSLQEN GELSPKSAEN WCRQQLMGMS RERSLQQYHQ TSDYQSAVRK YIEQAVGRAG RTSLKRKQIL LFVDSGLKEI LAEESRDPS LFSHEYVALV NKAKSAGKSI VEDRAVRRLF NLAQRNNKDG MLSIKALVHR LHNQPASKSD IQEWQDIRTQ L LRYPTVAF QPERFNRLYL QSMTKGYYRY QGNLDGDPNS FEFFDRVPYG DMVSEEDCSL ATLVQNQYVR PWFERKGFAC SW QKEANVM TPIMFTNIYK GALGEQAVEA VLTAFDFTFE EVPNSIYERF DNRVIFAGIE QPIWLDSKYW KHEGNESSEG YSS KIALVE EEFGPSKFIY VNALGDTSKP IRYLNSCFVE TSPQLAKVIE IPALIDDSNA DTNRTAVQEL IKWLHHS

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

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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

Concentration1.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris hydrochloride
350.0 mMNaClSodium chloride
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 59.98 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2023352
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1.) / Number images used: 184227
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1.)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: Initial model was generated with alpha fold
Output model

PDB-9ezx:
Vibrio cholerae DdmD apo complex

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