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- EMDB-50091: Vibrio cholerae DdmD-DdmE holo complex -

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Basic information

Entry
Database: EMDB / ID: EMD-50091
TitleVibrio cholerae DdmD-DdmE holo complex
Map dataUnsharpened EM volume
Sample
  • Complex: DdmD-DdmE Holocomplex
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • Protein or peptide: Vibrio cholerae DdmE
    • DNA: 14 nucleotide DNA guide with terminal 5' phosphate
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
KeywordsHelicase / Nuclease / Complex / Effector / Argonaute / IMMUNE SYSTEM
Function / homologyHelicase/UvrB N-terminal domain-containing protein / Uncharacterized protein
Function and homology information
Biological speciesVibrio cholerae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.56 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) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50091.png

Downloads & links

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Map

FileDownload / File: emd_50091.map.gz / Format: CCP4 / Size: 713.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened EM volume
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 572 pix.
= 371.8 Å		0.65 Å/pix.
x 572 pix.
= 371.8 Å		0.65 Å/pix.
x 572 pix.
= 371.8 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: 0.0257
Minimum - Maximum-0.07145727 - 0.19073372
Average (Standard dev.)-0.000052482726 (±0.0033284551)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions572572572
Spacing572572572
CellA=B=C: 371.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50091_msk_1.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 : DdmD-DdmE Holocomplex

EntireName: DdmD-DdmE Holocomplex
Components
  • Complex: DdmD-DdmE Holocomplex
    • Protein or peptide: Helicase/UvrB N-terminal domain-containing protein
    • Protein or peptide: Vibrio cholerae DdmE
    • DNA: 14 nucleotide DNA guide with terminal 5' phosphate
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DdmD-DdmE Holocomplex

SupramoleculeName: DdmD-DdmE Holocomplex / 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: 1 / 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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Macromolecule #2: Vibrio cholerae DdmE

MacromoleculeName: Vibrio cholerae DdmE / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 79.468156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMVTPQLE PSSQGPLSTL IEQISIDTDW VDRSFAIYCV SYKGIDFSER PKRLVTLASE TYKSGSVYCL VKGANKEACY WVLLPKDSK LDLKDTSLAI KPSSAAELPT WQLARLLIKA IPKVLSGTMP EIKRFESEGL YYLVKSKKLP KDHSGYELTT V EIDLAPCA ...String:
SNAMVTPQLE PSSQGPLSTL IEQISIDTDW VDRSFAIYCV SYKGIDFSER PKRLVTLASE TYKSGSVYCL VKGANKEACY WVLLPKDSK LDLKDTSLAI KPSSAAELPT WQLARLLIKA IPKVLSGTMP EIKRFESEGL YYLVKSKKLP KDHSGYELTT V EIDLAPCA ALGFKQTLSM GTKTFSPLSW FTLENGEVQK KARFATRYQL DDVGKLVSKS IKGDYIKKPL YSNAKNRIQA ID ITKESYS GFQLSKVGIL EQFMQDLKQA YGDSVSVKLQ RIPGEKHRFV SDTIVKNHYV GLFDALKEHR LVICDLTENQ DTD AALTLL HGIEHLDINA EIAEVPIRGA LNILIVGNKD TYKSDEEDPY QVYRKKYQDT VFQSCYPERL WNRQGQPNRH VVEV LLKEL LIKLEVHTRK HLIEYPSGPE RCVYYMPQRP KDESSEVRDE PWPVYASKLV GDEWQYTQAT QEELEDIELD LGNDK RHVF HGFERSPVIY WPETGDYAIF IDTGIQMLPE FEAVAERLRE LKEGRSQDVP IALLAQFIEE NPESKVINKL RAILSE WDD VAPLPFDEFS TIAYKSSDEK QFYDWLREQG FFLKTSIRGQ SEGFFNASLG FFYNREQGMY FAGGKGSPQS KIETFSH LY LIKHSFDALP EEVENLFDVY HLRHRLPTVT PYPFKHLREY VEMQRFRS

UniProtKB: Uncharacterized protein

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

MacromoleculeName: 14 nucleotide DNA guide with terminal 5' phosphate / type: dna / ID: 3
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.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.567998 KDa
SequenceString:
(DA)(DT)(DA)(DA)(DT)(DT)(DC)(DT)(DG)(DT) (DC)(DC)(DA)(DG)(DA)

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Macromolecule #4: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.341029 KDa
SequenceString: (DA)(DT)(DC)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DT)(DC)(DG)(DT)(DT)(DG)(DT)(DC)(DG)(DT) (DC)(DG)(DT)(DC)(DT)(DG)(DG)(DA)(DC) (DA)(DG)(DA)(DA)(DT)(DT)(DA)(DC)(DT)(DG) (DG) (DT)(DA)(DA)(DC)(DT)(DA) ...String:
(DA)(DT)(DC)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DT)(DC)(DG)(DT)(DT)(DG)(DT)(DC)(DG)(DT) (DC)(DG)(DT)(DC)(DT)(DG)(DG)(DA)(DC) (DA)(DG)(DA)(DA)(DT)(DT)(DA)(DC)(DT)(DG) (DG) (DT)(DA)(DA)(DC)(DT)(DA)(DC)(DG) (DG)(DT)(DG)(DG)(DA)(DC)(DA)(DA)(DT)(DG) (DC)(DT) (DA)(DC)(DC)(DT)(DT)(DA)

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Macromolecule #5: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.305029 KDa
SequenceString: (DT)(DA)(DA)(DG)(DG)(DT)(DA)(DG)(DC)(DA) (DT)(DT)(DG)(DT)(DC)(DC)(DA)(DC)(DC)(DG) (DT)(DA)(DG)(DT)(DT)(DA)(DC)(DC)(DA) (DG)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DC)(DG) ...String:
(DT)(DA)(DA)(DG)(DG)(DT)(DA)(DG)(DC)(DA) (DT)(DT)(DG)(DT)(DC)(DC)(DA)(DC)(DC)(DG) (DT)(DA)(DG)(DT)(DT)(DA)(DC)(DC)(DA) (DG)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DC)(DG)(DA)(DC) (DG)(DA)(DC)(DA)(DA)(DC)(DG)(DA)(DT)(DC) (DA)(DG) (DA)(DT)(DA)(DG)(DA)(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

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris hydrochloride
225.0 mMNaClSodium chloride
2.5 mMDesthiobiotinDesthiobiotin
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.99 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: 1283702
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1.) / Number images used: 229173
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-9ezy:
Vibrio cholerae DdmD-DdmE holo complex

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