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- PDB-8ti9: CryoEM structure of octamer assembly of Shedu nuclease domain fro... -

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Basic information

Entry
Database: PDB / ID: 8ti9
TitleCryoEM structure of octamer assembly of Shedu nuclease domain from Bacillus cereus
ComponentsShedu protein SduA
KeywordsDNA BINDING PROTEIN / Shedu / DUF4263 / Bacterial defense systems / Nuclease / Anti-plasmid defense system / PD-(D/E)XK nuclease / Whirly domain / Two-component signaling
Function / homologyProtein of unknown function DUF4263 / : / Shedu protein SduA, C-terminal / Shedu protein SduA, N-terminal / nuclease activity / defense response to virus / Shedu protein SduA
Function and homology information
Biological speciesBacillus cereus B4264 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsGu, Y. / Corbett, K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R35 GM144121 United States
CitationJournal: Mol Cell / Year: 2025
Title: Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains.
Authors: Yajie Gu / Huan Li / Amar Deep / Eray Enustun / Dapeng Zhang / Kevin D Corbett /
Abstract: Prokaryotes possess diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here, we reveal the structural basis for activation of Bacillus cereus Shedu. Two ...Prokaryotes possess diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here, we reveal the structural basis for activation of Bacillus cereus Shedu. Two cryoelectron microscopy structures of Shedu show that it switches between inactive and active states through conformational changes affecting active-site architecture, which are controlled by the protein's N-terminal domain (NTD). We find that B. cereus Shedu cleaves near DNA ends with a 3' single-stranded overhang, likely enabling it to specifically degrade the DNA injected by certain bacteriophages. Bioinformatic analysis of Shedu homologs reveals a conserved nuclease domain with remarkably diverse N-terminal regulatory domains: we identify 79 distinct NTD types falling into eight broad classes, including those with predicted nucleic acid binding, enzymatic, and other activities. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse NTDs that likely respond to a range of signals.
History
DepositionJul 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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
E: Shedu protein SduA
F: Shedu protein SduA
G: Shedu protein SduA
H: Shedu protein SduA
A: Shedu protein SduA
B: Shedu protein SduA
C: Shedu protein SduA
D: Shedu protein SduA


Theoretical massNumber of molelcules
Total (without water)209,8948
Polymers209,8948
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Shedu protein SduA / Putative nuclease SduA


Mass: 26236.760 Da / Num. of mol.: 8 / Mutation: E264A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus B4264 (bacteria) / Gene: sduA, BCB4264_A0974 / Production host: Escherichia coli (E. coli) / References: UniProt: B7HFR2
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: Bacillus cereus Shedu delta_NL octamer / Type: COMPLEX
Details: Octamer assembly of Bacillus cereus Shedu nuclease domain, which truncates the N-terminal and linker region. Glutamic acid 264 is mutated to Alanine.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.216 MDa / Experimental value: YES
Source (natural)Organism: Bacillus cereus B4264 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: Prepared using deionized water and filtered strelized.
Buffer component
IDConc.NameFormulaBuffer-ID
1500 mMSodium ChlorideNaCl1
220 mMHEPESC8H18N2O4S1
31 mMDithiothreitolC4H10O2S21
45 mMMagnesium ChlorideMgCl21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Freshly collected from size-exclusion column
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.2.1CTF correction
7Coot0.9.6model fitting
12cryoSPARC4.2.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138326 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212736
ELECTRON MICROSCOPYf_angle_d0.35717314
ELECTRON MICROSCOPYf_dihedral_angle_d9.3214630
ELECTRON MICROSCOPYf_chiral_restr0.042068
ELECTRON MICROSCOPYf_plane_restr0.0032146

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