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- PDB-8ps5: Escherichia coli SduA complex bound to DNA -

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Basic information

Entry
Database: PDB / ID: 8ps5
TitleEscherichia coli SduA complex bound to DNA
Components
  • 40 nt DNA substrate
  • Shedu effector protein
KeywordsIMMUNE SYSTEM / SduA / Shedu / Prokaryotic immune system
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesEscherichia coli KTE10 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 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
E: 40 nt DNA substrate
F: 40 nt DNA substrate


Theoretical massNumber of molelcules
Total (without water)214,4856
Polymers214,4856
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area32030 Å2
ΔGint-173 kcal/mol
Surface area81260 Å2
MethodPISA

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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: SduA / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: DNA chain 40 nt DNA substrate


Mass: 12311.926 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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

Component
IDNameTypeEntity IDParent-IDSource
1Tetrameric SduA complexCOMPLEXall0MULTIPLE SOURCES
2SduACOMPLEX#11RECOMBINANT
3DNACOMPLEX#21RECOMBINANT
Molecular weightValue: 0.194 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli KTE10 (bacteria)1169330
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl pH 8, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClNH2C(CH2OH)3HCl1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 2.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
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: 60.01 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 9799

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7Coot0.9.2model fitting
9PHENIXmodel refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1977096
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 260089 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515338
ELECTRON MICROSCOPYf_angle_d0.94721068
ELECTRON MICROSCOPYf_dihedral_angle_d21.4892570
ELECTRON MICROSCOPYf_chiral_restr0.0552358
ELECTRON MICROSCOPYf_plane_restr0.0082420

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