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- PDB-8tia: CryoEM structure of locally-refined tetramer of Shedu nuclease do... -

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Basic information

Entry
Database: PDB / ID: 8tia
TitleCryoEM structure of locally-refined tetramer 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: 2.77 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: Database references / Structure summary / Category: citation / citation_author / 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
Revision 1.2Feb 19, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3May 28, 2025Group: Data collection / Category: em_software / Item: _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Shedu protein SduA
B: Shedu protein SduA
C: Shedu protein SduA
D: Shedu protein SduA


Theoretical massNumber of molelcules
Total (without water)104,9474
Polymers104,9474
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.: 4 / 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: Shedu nuclease domain in an active form, which lacks N-terminal domains. A tetramer copy of the overall octamer assembly complex.
Type: COMPLEX
Details: This is a local refinement of a tetramer copy of the octamer assembly complex
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus cereus B4264 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
8PHENIXmodel refinement
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 331076 / Algorithm: FOURIER SPACE
Details: Local refinement using cryoSPARC, with NU refinement option enabled.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026365
ELECTRON MICROSCOPYf_angle_d0.3188653
ELECTRON MICROSCOPYf_dihedral_angle_d8.1732313
ELECTRON MICROSCOPYf_chiral_restr0.0381033
ELECTRON MICROSCOPYf_plane_restr0.0021073

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