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Yorodumi- EMDB-25567: Cryo-EM structure of OmpK36-TraN mating pair stabilization protei... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25567 | |||||||||
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Title | Cryo-EM structure of OmpK36-TraN mating pair stabilization proteins from carbapenem-resistant Klebsiella pneumoniae | |||||||||
Map data | Whole OmpK36-TraN map | |||||||||
Sample |
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Function / homology | Function and homology information porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane Similarity search - Function | |||||||||
Biological species | Klebsiella pneumoniae (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.7 Å | |||||||||
Authors | Beltran LC / Seddon C / Beis K / Frankel G / Egelman EH | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Nat Microbiol / Year: 2022 Title: Mating pair stabilization mediates bacterial conjugation species specificity. Authors: Wen Wen Low / Joshua L C Wong / Leticia C Beltran / Chloe Seddon / Sophia David / Hok-Sau Kwong / Tatiana Bizeau / Fengbin Wang / Alejandro Peña / Tiago R D Costa / Bach Pham / Min Chen / ...Authors: Wen Wen Low / Joshua L C Wong / Leticia C Beltran / Chloe Seddon / Sophia David / Hok-Sau Kwong / Tatiana Bizeau / Fengbin Wang / Alejandro Peña / Tiago R D Costa / Bach Pham / Min Chen / Edward H Egelman / Konstantinos Beis / Gad Frankel / Abstract: Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of ...Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraN interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25567.map.gz | 55.3 MB | EMDB map data format | |
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Header (meta data) | emd-25567-v30.xml emd-25567.xml | 21.5 KB 21.5 KB | Display Display | EMDB header |
Images | emd_25567.png | 228.1 KB | ||
Others | emd_25567_additional_1.map.gz emd_25567_additional_2.map.gz emd_25567_additional_3.map.gz | 2.9 MB 3.2 MB 149 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25567 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25567 | HTTPS FTP |
-Related structure data
Related structure data | 7sziMC M: atomic model generated by this map C: citing same article (ref.) |
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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_25567.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Whole OmpK36-TraN map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: TraN loop only
File | emd_25567_additional_1.map | ||||||||||||
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Annotation | TraN loop only | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: OmpK36 Barrels only
File | emd_25567_additional_2.map | ||||||||||||
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Annotation | OmpK36 Barrels only | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: TraN Flexible Tail
File | emd_25567_additional_3.map | ||||||||||||
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Annotation | TraN Flexible Tail | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Conjugal mating pair stabilizing complex OmpK36 and TraN
Entire | Name: Conjugal mating pair stabilizing complex OmpK36 and TraN |
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Components |
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-Supramolecule #1: Conjugal mating pair stabilizing complex OmpK36 and TraN
Supramolecule | Name: Conjugal mating pair stabilizing complex OmpK36 and TraN type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all Details: OmpK36WT ATCC43816 serially passaged in vitro on Rifampicin containing LB plates to 100mcg/ml followed by two passages in BALB/c mice. Mature TraN gene D28 to Q651 from pKpQIL was sub-cloned ...Details: OmpK36WT ATCC43816 serially passaged in vitro on Rifampicin containing LB plates to 100mcg/ml followed by two passages in BALB/c mice. Mature TraN gene D28 to Q651 from pKpQIL was sub-cloned into the pTAMAHISTEV vector with an N-terminal His7-tag and a tobacco etch virus TEV cleavage site using the NcoI and XhoI restriction enzyme sites. The construct was transformed into E. coli C43 DE3 competent cells and expressed in Terrific Broth TB medium Formedium supplemented with 100 microgram/mL ampicillin. |
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Source (natural) | Organism: Klebsiella pneumoniae (bacteria) / Strain: ICC8001 |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pTAMAHISTEV |
Molecular weight | Experimental: 120 KDa |
-Macromolecule #1: OmpK36
Macromolecule | Name: OmpK36 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Klebsiella pneumoniae (bacteria) / Strain: ICC8001 |
Molecular weight | Theoretical: 38.058918 KDa |
Sequence | String: GAEIYNKDGN KLDLYGKIDG LHYFSDDKSV DGDQTYMRVG VKGETQINDQ LTGYGQWEYN VQANNTESSS DQAWTRLAFA GLKFGDAGS FDYGRNYGVV YDVTSWTDVL PEFGGDTYGS DNFLQSRANG VATYRNSDFF GLVDGLNFAL QYQGKNGSVS G EGATNNGR ...String: GAEIYNKDGN KLDLYGKIDG LHYFSDDKSV DGDQTYMRVG VKGETQINDQ LTGYGQWEYN VQANNTESSS DQAWTRLAFA GLKFGDAGS FDYGRNYGVV YDVTSWTDVL PEFGGDTYGS DNFLQSRANG VATYRNSDFF GLVDGLNFAL QYQGKNGSVS G EGATNNGR GWSKQNGDGF GTSLTYDIWD GISAGFAYSH SKRTDEQNSV PALGRGDNAE TYTGGLKYDA NNIYLASQYT QT YNATRAG SLGFANKAQN FEVVAQYQFD FGLRPSVAYL QSKGKDLERG YGDQDILKYV DVGATYYFNK NMSTYVDYKI NLL DDNSFT RNAGISTDDV VALGLVYQF |
-Macromolecule #2: TraN
Macromolecule | Name: TraN / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Klebsiella pneumoniae (bacteria) / Strain: ST 258 |
Molecular weight | Theoretical: 906.964 Da |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: CSGGQNTHC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.033 mg/mL | ||||||||||||
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Buffer | pH: 7.8 Component:
Details: SEC Buffer | ||||||||||||
Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000 |
Specialist optics | Energy filter - Slit width: 10 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number real images: 13688 / Average exposure time: 4.78 sec. / Average electron dose: 50.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 13780567 |
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CTF correction | Software - Name: cryoSPARC (ver. 3.2.0) |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 598537 |
-Atomic model buiding 1
Refinement | Space: REAL |
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Output model | PDB-7szi: |