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- EMDB-27480: CryoEM structure of JetABC (head construct) from Pseudomonas aeru... -

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Basic information

Entry
Database: EMDB / ID: EMD-27480
TitleCryoEM structure of JetABC (head construct) from Pseudomonas aeruginosa PA14
Map data
Sample
  • Complex: Reconstituted JetABC head construct
    • Protein or peptide: JetA
    • Protein or peptide: JetC
    • Protein or peptide: JetB
KeywordsWadjet / Bacterial defense systems / JetA / JetB / JetC / Anti-plasmid defense system / EptA / EptB / EptC / MksB / MksE / MksF / SMC / DNA BINDING PROTEIN-DNA complex
Function / homologyWadjet protein JetB / Domain of unknown function (DUF4194) / Protein of unknown function DUF3375 / Protein of unknown function (DUF3375) / P-loop containing nucleoside triphosphate hydrolase / DUF3375 domain-containing protein / DUF4194 domain-containing protein / ATP synthase
Function and homology information
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 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_27480.png

Downloads & links

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Map

FileDownload / File: emd_27480.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.18394881 - 0.5359808
Average (Standard dev.)0.0007681129 (±0.0154705485)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: local refinement of the JetB dimer.

Fileemd_27480_additional_1.map
Annotationlocal refinement of the JetB dimer.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: local refinement of the JetB dimer.

Fileemd_27480_additional_2.map
Annotationlocal refinement of the JetB dimer.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: local refinement of the JetB dimer.

Fileemd_27480_additional_3.map
Annotationlocal refinement of the JetB dimer.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27480_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27480_half_map_2.map
Projections & Slices
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Sample components

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Entire : Reconstituted JetABC head construct

EntireName: Reconstituted JetABC head construct
Components
  • Complex: Reconstituted JetABC head construct
    • Protein or peptide: JetA
    • Protein or peptide: JetC
    • Protein or peptide: JetB

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Supramolecule #1: Reconstituted JetABC head construct

SupramoleculeName: Reconstituted JetABC head construct / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: A Pseudomonas aeruginosa protein complex involved in anti-plasmid defense system.
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 369 KDa

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Macromolecule #1: JetA

MacromoleculeName: JetA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / 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 #2: JetC

MacromoleculeName: JetC / type: protein_or_peptide / ID: 2
Details: JetC head domain is constructed through a glycine serine link replacing two long coiled-coils. Additional mutations are: E1022Q, L783A.,JetC head domain is constructed through a glycine ...Details: JetC head domain is constructed through a glycine serine link replacing two long coiled-coils. Additional mutations are: E1022Q, L783A.,JetC head domain is constructed through a glycine serine link replacing two long coiled-coils. Additional mutations are: E1022Q, L783A.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 70.675375 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 QDKQASGSSG SSGSSGQAKT LGE KLGDQK TELAKRMSDA LKADTGALAE VGRELVDVPR YLERLRVLTE EALPEKLKRF LEYLNRSSDD GVTQLLSYID HEVS MIEER LDDLNSTMQR VDFQPGRYLR LVAKKVIHES LRTLQHAQRQ LNSARFIDDE GESHYKALQA LVGLLKDACE HSRNQ GAKA LLDPRFRLEF AVSVIDREGN NLIETRTGSQ GGSGGEKEII ASYVLTASLS YALCPDGSSR PLFGTIVLDQ AFSRSS HAV AGRIIAALRE FGLHAVFITP NKEMRLLRHH TRSAVVVHRR GVESSLVSLS WEALDEHHQQ RIRAMHEVAH

UniProtKB: ATP synthase, ATP synthase

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Macromolecule #3: JetB

MacromoleculeName: JetB / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Molecular weightTheoretical: 27.537037 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAGIFDRIAG ASGADETELT AEPMALDDGM DGEQPAMSAN IQVDERRTPQ RVREAVQEML KYGLLEESHK PNLYRSALTN IEVVDRILE PLDLAMGVDE VRGLVFVTVR QGEVAEQDDW SHPLVRRQRL NLEQSLLIAI LRQHFIAYEQ ESGTGASQAL V AVDELIPQ ...String:
MAGIFDRIAG ASGADETELT AEPMALDDGM DGEQPAMSAN IQVDERRTPQ RVREAVQEML KYGLLEESHK PNLYRSALTN IEVVDRILE PLDLAMGVDE VRGLVFVTVR QGEVAEQDDW SHPLVRRQRL NLEQSLLIAI LRQHFIAYEQ ESGTGASQAL V AVDELIPQ LQVYLGELGS EAKERNRIIT LLDQLKGHGL VSALDAHDRV IIRPIITHLA NPENLQALVV WLREQVEGAV TP AAGGEED EA

UniProtKB: DUF4194 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
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: 54.1 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: 165000
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: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 130904
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-8dk1:
CryoEM structure of JetABC (head construct) from Pseudomonas aeruginosa PA14

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