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- PDB-8ps4: Escherichia coli SduA complex -

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Basic information

Entry
Database: PDB / ID: 8ps4
TitleEscherichia coli SduA complex
ComponentsShedu effector protein
KeywordsIMMUNE SYSTEM / SduA / Shedu / Prokaryotic immune system
Biological speciesEscherichia coli KTE10 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsLoeff, L. / Jinek, M.
Funding supportEuropean Union, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Shedu effector protein
B: Shedu effector protein
C: Shedu effector protein
D: Shedu effector protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,9106
Polymers189,8614
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, Size of the assembly was verified using SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Shedu effector protein / Shedu protein / DUF4263 domain-containing protein


Mass: 47465.273 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli KTE10 (bacteria) / Gene: ELX76_26215 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tetrameric SduA complex / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.194 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli KTE10 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClNH2C(CH2OH)3HCl1
2150 mMSodium chlorideNaCl1
35 mMMagnesium chlorideMgCl1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Samples was monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66.529 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5289

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7Coot0.9.2model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1096246
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202998 / Symmetry type: POINT

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