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- EMDB-25913: CryoEM structure of JetD from Pseudomonas aeruginosa -

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Basic information

Entry
Database: EMDB / ID: EMD-25913
TitleCryoEM structure of JetD from Pseudomonas aeruginosa
Map data
Sample
  • Complex: JetD
    • Protein or peptide: JetD
KeywordsWadjet / Bacterial defense systems / JetD / Anti-plasmid defense system / EptD / MksG / Toprim domain / Nuclease / Topoisomerase / DNA BINDING PROTEIN
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) / DUF3322 and DUF2220 domain-containing protein
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDeep A / Gu Y
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25913.png

Downloads & links

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Map

FileDownload / File: emd_25913.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 448 pix.
= 291.2 Å		0.65 Å/pix.
x 448 pix.
= 291.2 Å		0.65 Å/pix.
x 448 pix.
= 291.2 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
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Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.10068573 - 0.2679501
Average (Standard dev.)-0.00018519773 (±0.0049183723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 291.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #6

Fileemd_25913_additional_1.map
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Sample components

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Entire : JetD

EntireName: JetD
Components
  • Complex: JetD
    • Protein or peptide: JetD

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Supramolecule #1: JetD

SupramoleculeName: JetD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: A Pseudomonas aeruginosa protein.
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 85.6 kDa/nm

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Macromolecule #1: JetD

MacromoleculeName: JetD / type: protein_or_peptide / ID: 1
Details: Pseudomonas aeruginosa PA14 JetD protein with N-terminal TEV cleavable 6XHis tag.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria) / Strain: UCBPP-PA14
Molecular weightTheoretical: 45.132164 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKSSHHHHHH ENLYFQSNAK SPTDIGRNLA RQWQRSSVRL ERLLNPGSWP QSLNIGKPSA RLFTDNIQAV LRHVENWRSV NVGKVDWEP VSYRAGLAPI SLPMRWHLRS PSEWIAASDD PRVSQEYAQL EYLVEQVDSA FHTLLVAQRS LWLTRTPEEV V ATAQLATQ ...String:
MKSSHHHHHH ENLYFQSNAK SPTDIGRNLA RQWQRSSVRL ERLLNPGSWP QSLNIGKPSA RLFTDNIQAV LRHVENWRSV NVGKVDWEP VSYRAGLAPI SLPMRWHLRS PSEWIAASDD PRVSQEYAQL EYLVEQVDSA FHTLLVAQRS LWLTRTPEEV V ATAQLATQ LAPGCAQGRP LRLLAEHGVD TKFFERNATL LTKLLDERFE GAASEQGLTT FLDAFEESSH WVLVVPLQPG LL PFKRLRL TTSELAETPL PGSRLLVVEN EQCVHLLPET LPDTLAVLGA GLDLHWLASA HLAGKQIGYW GDMDTWGLLM LAR ARLHQP AVEALLMEQE LFEQHSQGNT VVEPAKALES APPGLLADEA DFYRYLLVQE RGRLEQEYLP KAQVELAIRK WAR

UniProtKB: DUF3322 and DUF2220 domain-containing protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.10 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
20.0 mMC4H11NO3tris(hydroxymethyl)aminomethane
2.0 mMHOCH2CH2SHB-mercaptoethanol

Details: Prepared using deionized water and filtered strelized.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51336
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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