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- EMDB-27482: CryoEM structure of Pseudomonas aeruginosa PA14 JetC ATPase domai... -

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Basic information

Entry
Database: EMDB / ID: EMD-27482
TitleCryoEM structure of Pseudomonas aeruginosa PA14 JetC ATPase domain bound to DNA and cWHD domain of JetA
Map data
Sample
  • Complex: JetC head ATPase domain in complex with DNA, and cWHD of JetA
    • Protein or peptide: JetC
    • Protein or peptide: JetA
    • DNA: DNA (26-MER)
    • DNA: DNA (26-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsWadjet / Bacterial defense systems / JetA / JetC / Anti-plasmid defense system / EptA / EptC / MksB / MksF / SMC / DNA BINDING PROTEIN-DNA complex
Function / homologyProtein of unknown function DUF3375 / Protein of unknown function (DUF3375) / P-loop containing nucleoside triphosphate hydrolase / DUF3375 domain-containing protein / ATP synthase
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsDeep A / Gu Y / Gao Y / Ego K / Herzik M / Zhou H / Corbett K
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
DepositionJul 1, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27482.png

Downloads & links

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Map

FileDownload / File: emd_27482.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.33449867 - 0.77822816
Average (Standard dev.)0.0006912143 (±0.017972255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27482_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27482_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : JetC head ATPase domain in complex with DNA, and cWHD of JetA

EntireName: JetC head ATPase domain in complex with DNA, and cWHD of JetA
Components
  • Complex: JetC head ATPase domain in complex with DNA, and cWHD of JetA
    • Protein or peptide: JetC
    • Protein or peptide: JetA
    • DNA: DNA (26-MER)
    • DNA: DNA (26-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: JetC head ATPase domain in complex with DNA, and cWHD of JetA

SupramoleculeName: JetC head ATPase domain in complex with DNA, and cWHD of JetA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: JetC

MacromoleculeName: JetC / type: protein_or_peptide / ID: 1 / Details: The sequence contains mutation: E1022Q. / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 126.68318 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKSSHHHHHH ENLYFQSNAK QALLWRDSET FILTSIELYN WGGFQGYHRA EIDPSGTAVI GPTGSGKTTL VDALMTLLCA NPRYNLAST GGHESDRDLV SYVRGVTGPG DGGVEQSHIA RQGKTVTAIA ATLERDGAQV RLGAVLWFEG TSSSASDLKK L WLLSESPE ...String:
MKSSHHHHHH ENLYFQSNAK QALLWRDSET FILTSIELYN WGGFQGYHRA EIDPSGTAVI GPTGSGKTTL VDALMTLLCA NPRYNLAST GGHESDRDLV SYVRGVTGPG DGGVEQSHIA RQGKTVTAIA ATLERDGAQV RLGAVLWFEG TSSSASDLKK L WLLSESPE QTLEHWLSQH HAGGMRALRQ MEKDGMGIWP YPSKKAFLAR LRDYFEVGEN AFTLLNRAAG LKQLNSIDEI FR ELVLDDR SAFERAAEVA SSFDDLTDIH RELETARKQQ RSLQPVADGW ERYRALQEQL QDKQALEGIL PVWFAEQGYR LWL AETNRL EKEHKQAELD QAQCRSQLEI QKGVVDQHRQ RYLRVGGAGI DQLRGRIADW VRECDKRRLK AEQYQRLAKG LGLA DELSA AALEENQQQI AARLEILAQQ TTDARQKAFD AGLVQQELNG RLQSLQQERA EVERRPGSNL PGHFHAFRGD LAQEL GVDE SALPFVAELV QVKPEELAWR GAIERAIGSH RLRILVPQGS SQAALRWVNQ RHNRLHVRLL EVKEPSSRPV FFDDGF TRK LTFKEHPYRE AVKALLADND RHCVESTEQL RHTPHAMTAQ GLMSGKERFF DKQDQKRLDE DWLTGFDNRD RLAFLAE QI REVNEQLVPA KLALDAAQGD VGQLESQASL LQRIEELQFD DIDRPGAERQ LQSLRTQLDT LTRPDSNLAV IKAELDQA E ALRESLDQQL QRLIEQCVQL KTQFDQAASA TRKAYRGAEK GLSDTQRELA QAHFPILSTD DLGDIDELER KHTRELQGQ LKTLGEKLGD QKTELAKRMS DALKADTGAL AEVGRELVDV PRYLERLRVL TEEALPEKLK RFLEYLNRSS DDGVTQLLSY IDHEVSMIE ERLDDLNSTM QRVDFQPGRY LRLVAKKVIH ESLRTLQHAQ RQLNSARFID DEGESHYKAL QALVGLLKDA C EHSRNQGA KALLDPRFRL EFAVSVIDRE GNNLIETRTG SQGGSGGEKE IIASYVLTAS LSYALCPDGS SRPLFGTIVL DQ AFSRSSH AVAGRIIAAL REFGLHAVFI TPNKEMRLLR HHTRSAVVVH RRGVESSLVS LSWEALDEHH QQRIRAMHEV AH

UniProtKB: ATP synthase

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Macromolecule #2: JetA

MacromoleculeName: JetA / type: protein_or_peptide / ID: 2
Details: cWHD domain of JetA observed in complex with dimeric JetC.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 59.2335 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKSSHHHHHH ENLYFQSNAE ESAQQRSERY VSARSQHPAW LLLASRRAPL VLGCLRTLFE RAHDGIPMED ALQALSEMLA AYASQELYE IDPDATHLQA GRELREWIKR RLVVEREGRI YATDALESAI QFVDSLDSRI MTSTASRLSV VQREIENLET G LNPSPTGR ...String:
MKSSHHHHHH ENLYFQSNAE ESAQQRSERY VSARSQHPAW LLLASRRAPL VLGCLRTLFE RAHDGIPMED ALQALSEMLA AYASQELYE IDPDATHLQA GRELREWIKR RLVVEREGRI YATDALESAI QFVDSLDSRI MTSTASRLSV VQREIENLET G LNPSPTGR IASLRRRIQD LEHELARVEA GHVDVLDEAQ AIEGMREVYN LATSLRADFR RVEDSWREAD RALRHSIISE QS HRGEIVD RLLDGQDALL NTPEGRVFES FQQQLRQSAE LEVMRERLRT ILRHPAVPKA LNRPQQRELR WLALRLVRES QAV LQARAR SERDVRGFMK TGLAAEHHRV GQLLNDFFNL ALSVDWQRQS ERRKPACLPP VGVAITGVPA IERLRFKTLD DDDA GELDL SLKPAGLEQI DDDFWDAFDG LDREALIHDT LAVLVEQGRP VSLGELASLL PPAHDLETFA LWLAMAREAG IEVLT EERQ FVELVDEDEQ RWGFNLPYVG LDHEALKDID WEL

UniProtKB: DUF3375 domain-containing protein

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Macromolecule #3: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 3
Details: Below mentioned hybridized oligonucleotides were used in the sample preparation. Considering the DNA binding is purely based on the electrostatic charge: polyA/polyT chain was used to ...Details: Below mentioned hybridized oligonucleotides were used in the sample preparation. Considering the DNA binding is purely based on the electrostatic charge: polyA/polyT chain was used to generate the model. JetABC_cryo_Fwd: GTGATAGTTAGAAACGTAATTGACTATAAAGATGATGACGATAAGTAGGATGTCTATAGACCAGG JetABC_cryo_Rev: CCTGGTCTATAGACATCCTACTTATCGTCATCATCTTTATAGTCAATTACGTTTCTAACTATCAC
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.864056 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #4: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 4
Details: Below mentioned hybridized oligonucleotides were used in the sample preparation. Considering the DNA binding is purely based on the electrostatic charge: polyA/polyT chain was used to ...Details: Below mentioned hybridized oligonucleotides were used in the sample preparation. Considering the DNA binding is purely based on the electrostatic charge: polyA/polyT chain was used to generate the model. JetABC_cryo_Fwd: GTGATAGTTAGAAACGTAATTGACTATAAAGATGATGACGATAAGTAGGATGTCTATAGACCAGG JetABC_cryo_Rev: CCTGGTCTATAGACATCCTACTTATCGTCATCATCTTTATAGTCAATTACGTTTCTAACTATCAC
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.098421 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 2 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
60.0 mMNaClSodium Chloride
30.0 mMC4H11NO3tris(hydroxymethyl)aminomethane
1.0 mMC9H16ClO6PTCEP
1.0 mMC10H16N5O12P3SATP gamma S
2.0 mMMgCl2Magnesium Chloride

Details: Prepared using deionized water and filtered sterilized.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
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) / Detector mode: COUNTING / Average electron dose: 50.1 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 / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 323026
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-8dk3:
CryoEM structure of Pseudomonas aeruginosa PA14 JetC ATPase domain bound to DNA and cWHD domain of JetA

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