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Yorodumi- EMDB-27481: CryoEM structure of Pseudomonas aeruginosa PA14 JetABC in an uncl... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27481 | |||||||||
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Title | CryoEM structure of Pseudomonas aeruginosa PA14 JetABC in an unclamped state trapped in ATP dependent dimeric form | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Wadjet / Bacterial defense systems / JetA / JetB / JetC / Anti-plasmid defense system / EptA / EptB / EptC / MksB / MksE / MksF / SMC / DNA BINDING PROTEIN-DNA complex | |||||||||
Function / homology | Wadjet 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 species | Pseudomonas aeruginosa PA14 (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.1 Å | |||||||||
Authors | Deep A / Gu Y / Gao Y / Ego K / Herzik M / Zhou H / Corbett K | |||||||||
Funding support | United States, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27481.map.gz | 108.8 MB | EMDB map data format | |
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Header (meta data) | emd-27481-v30.xml emd-27481.xml | 30 KB 30 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27481_fsc.xml | 12.7 KB | Display | FSC data file |
Images | emd_27481.png | 67.1 KB | ||
Filedesc metadata | emd-27481.cif.gz | 7.9 KB | ||
Others | emd_27481_additional_1.map.gz emd_27481_additional_2.map.gz emd_27481_additional_3.map.gz emd_27481_half_map_1.map.gz emd_27481_half_map_2.map.gz | 200.6 MB 200.6 MB 108.9 MB 200.3 MB 200.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27481 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27481 | HTTPS FTP |
-Validation report
Summary document | emd_27481_validation.pdf.gz | 902.4 KB | Display | EMDB validaton report |
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Full document | emd_27481_full_validation.pdf.gz | 902 KB | Display | |
Data in XML | emd_27481_validation.xml.gz | 21.5 KB | Display | |
Data in CIF | emd_27481_validation.cif.gz | 28.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27481 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27481 | HTTPS FTP |
-Related structure data
Related structure data | 8dk2MC 7tilC 8dk1C 8dk3C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_27481.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.4667 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: half map for focused refinement of JetC and DNA.
File | emd_27481_additional_1.map | ||||||||||||
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Annotation | half map for focused refinement of JetC and DNA. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: half map for focused refinement of JetC and DNA.
File | emd_27481_additional_2.map | ||||||||||||
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Annotation | half map for focused refinement of JetC and DNA. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: focused refinement of JetC and DNA, from over 170k particles.
File | emd_27481_additional_3.map | ||||||||||||
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Annotation | focused refinement of JetC and DNA, from over 170k particles. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_27481_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27481_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Reconstituted JetABC complex in the presence of DNA and ATP gamma S.
Entire | Name: Reconstituted JetABC complex in the presence of DNA and ATP gamma S. |
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Components |
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-Supramolecule #1: Reconstituted JetABC complex in the presence of DNA and ATP gamma S.
Supramolecule | Name: Reconstituted JetABC complex in the presence of DNA and ATP gamma S. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: Pseudomonas aeruginosa PA14 (bacteria) |
Molecular weight | Theoretical: 481 KDa |
-Macromolecule #1: JetA
Macromolecule | Name: JetA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa PA14 (bacteria) |
Molecular weight | Theoretical: 59.2335 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: JetC
Macromolecule | Name: JetC / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa PA14 (bacteria) |
Molecular weight | Theoretical: 126.68318 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #3: JetB
Macromolecule | Name: JetB / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa PA14 (bacteria) |
Molecular weight | Theoretical: 27.537037 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #4: DNA (26-MER)
Macromolecule | Name: 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 7.864056 KDa |
Sequence | String: (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) |
-Macromolecule #5: DNA (26-MER)
Macromolecule | Name: DNA (26-MER) / type: dna / ID: 5 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.098421 KDa |
Sequence | String: (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) |
-Macromolecule #6: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 2 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ChemComp-AGS: |
-Macromolecule #7: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.5 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
Details: Prepared using deionized water and filtered sterilized. | ||||||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.1 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL |
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Output model | PDB-8dk2: |