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- PDB-7til: CryoEM structure of JetD from Pseudomonas aeruginosa -

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Basic information

Entry
Database: PDB / ID: 7til
TitleCryoEM structure of JetD from Pseudomonas aeruginosa
ComponentsJetD
KeywordsDNA BINDING PROTEIN / Wadjet / Bacterial defense systems / JetD / Anti-plasmid defense system / EptD / MksG / Toprim domain / Nuclease / Topoisomerase
Function / homologyUncharacterised conserved protein UCP028408 / Wadjet protein JetD, C-terminal / Domain of unknown function DUF3322 / Wadjet protein JetD, C-terminal / Uncharacterized protein conserved in bacteria N-term (DUF3322) / Uncharacterized protein
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDeep, A. / Gu, Y. / Gao, Y. / Ego, K. / Herzik, M. / Zhou, H. / Corbett, K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 GM104141 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI148814 United States
CitationJournal: Mol Cell / Year: 2022
Title: The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.
Authors: Amar Deep / Yajie Gu / Yong-Qi Gao / Kaori M Ego / Mark A Herzik / Huilin Zhou / Kevin D Corbett /
Abstract: Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through ...Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.
History
DepositionJan 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation / citation_author
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 / _citation_author.name
Revision 1.2Nov 16, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: JetD
B: JetD


Theoretical massNumber of molelcules
Total (without water)90,2642
Polymers90,2642
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, Confirmed using Size-exclusion chromatography coupled with Multi-Angle Light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein JetD


Mass: 45132.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Pseudomonas aeruginosa PA14 JetD protein with N-terminal TEV cleavable 6XHis tag.
Source: (gene. exp.) Pseudomonas aeruginosa PA14 (bacteria) / Strain: UCBPP-PA14 / Gene: PA14_03285 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H2ZM47
Sequence detailsPseudomonas aeruginosa PA14 JetD protein with N-terminal TEV cleavable 6XHis tag.

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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: JetD / Type: COMPLEX / Details: A Pseudomonas aeruginosa protein. / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 85.6 kDa/nm / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa PA14 (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
1200 mMSodium ChlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneC4H11NO31
32 mMB-mercaptoethanolHOCH2CH2SH1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 3.3.1 / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51336 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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