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- EMDB-17845: Escherichia coli SduA complex bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-17845
TitleEscherichia coli SduA complex bound to DNA
Map dataSharpened map
Sample
  • Complex: Tetrameric SduA complex
    • Complex: SduA
      • Protein or peptide: Shedu effector protein
    • Complex: DNA
      • DNA: 40 nt DNA substrate
KeywordsSduA / Shedu / Prokaryotic immune system / IMMUNE SYSTEM
Biological speciesEscherichia coli KTE10 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsLoeff L / Jinek M / Asanovic I / Boneberg F / Pfleiderer MM / Ferdigg A / Ackle F / Martinez J
Funding supportEuropean Union, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)820150European Union
H2020 Marie Curie Actions of the European Commission845268European Union
CitationJournal: Cell / Year: 2025
Title: DNA end sensing and cleavage by the Shedu anti-phage defense system.
Authors: Luuk Loeff / Alexander Walter / Gian Tizio Rosalen / Martin Jinek /
Abstract: The detection of molecular patterns associated with invading pathogens is a hallmark of innate immune systems. Prokaryotes deploy sophisticated host defense mechanisms in innate anti-phage immunity. ...The detection of molecular patterns associated with invading pathogens is a hallmark of innate immune systems. Prokaryotes deploy sophisticated host defense mechanisms in innate anti-phage immunity. Shedu is a single-component defense system comprising a putative nuclease SduA. Here, we report cryoelectron microscopy (cryo-EM) structures of apo- and double-stranded DNA (dsDNA)-bound tetrameric SduA assemblies, revealing that the N-terminal domains of SduA form a clamp that recognizes free DNA ends. End binding positions the DNA over the PD-(D/E)XK nuclease domain, resulting in dsDNA nicking at a fixed distance from the 5' end. The end-directed DNA nicking activity of Shedu prevents propagation of linear DNA in vivo. Finally, we show that phages escape Shedu immunity by suppressing their recombination-dependent DNA replication pathway. Taken together, these results define the antiviral mechanism of Shedu systems, underlining the paradigm that recognition of pathogen-specific nucleic acid structures is a conserved feature of innate immunity across all domains of life.
History
DepositionJul 13, 2023-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17845.png

Downloads & links

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Map

FileDownload / File: emd_17845.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 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: 0.0799
Minimum - Maximum-0.48582065 - 0.727136
Average (Standard dev.)0.0005461775 (±0.019926209)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17845_msk_1.map
Projections & Slices
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Additional map: #1

Fileemd_17845_additional_1.map
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Half map: #2

Fileemd_17845_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_17845_half_map_2.map
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Sample components

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Entire : Tetrameric SduA complex

EntireName: Tetrameric SduA complex
Components
  • Complex: Tetrameric SduA complex
    • Complex: SduA
      • Protein or peptide: Shedu effector protein
    • Complex: DNA
      • DNA: 40 nt DNA substrate

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Supramolecule #1: Tetrameric SduA complex

SupramoleculeName: Tetrameric SduA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 194 KDa

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Supramolecule #2: SduA

SupramoleculeName: SduA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli KTE10 (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Shedu effector protein

MacromoleculeName: Shedu effector protein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli KTE10 (bacteria)
Molecular weightTheoretical: 47.465273 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMLQFSFV SNDVVMTYDG DSGEQIIWVW ESLNKFQTVC ISRIFNFQLQ DLRNPPSTVQ DFNDYEYSFN FGTLNNEYIT VPGRILSIN RDVLIHKSIK LERKVFASER NVSIFGRLSK LLDHTNPIII GGDKPEAIPK SVFQELQSKF PNTGELDRYA N ARVHAILA ...String:
SNAMLQFSFV SNDVVMTYDG DSGEQIIWVW ESLNKFQTVC ISRIFNFQLQ DLRNPPSTVQ DFNDYEYSFN FGTLNNEYIT VPGRILSIN RDVLIHKSIK LERKVFASER NVSIFGRLSK LLDHTNPIII GGDKPEAIPK SVFQELQSKF PNTGELDRYA N ARVHAILA GYLDGMKDAR ERYEHYLNRK TVIRKTDKLD LEVLNKLEIE KYTLIRDIIQ DALNNKTNLS EDDWQSLMIP FI TLLFPKY IKVLEKVKIF DYYSNPSAKT NRFIDIALVD ANGNLDIIEV KKPFDDKILR KTPYRDNYIP TSELSGGIMQ AEK YIFHLS KWGVKGEKEL TNAYKNSLPA GMCIRISNPK AIIIVGRDQI ANGNMTDGQL LDFEIIKRKY ANMIDILTYD DLLR RLNNT IEALKG

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Macromolecule #2: 40 nt DNA substrate

MacromoleculeName: 40 nt DNA substrate / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.311926 KDa
SequenceString:
(DC)(DT)(DA)(DG)(DT)(DG)(DC)(DA)(DT)(DC) (DT)(DG)(DA)(DA)(DT)(DC)(DG)(DT)(DC)(DA) (DT)(DG)(DA)(DC)(DG)(DA)(DT)(DT)(DC) (DA)(DG)(DA)(DT)(DG)(DC)(DA)(DC)(DT)(DA) (DG)

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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

Concentration2.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMNH2C(CH2OH)3HClTris-HCl
150.0 mMNaClSodium chloride

Details: 20 mM Tris-HCl pH 8, 150 mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277.15 K / 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) / Number real images: 9799 / Average electron dose: 60.01 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: 1977096
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 260089
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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