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- PDB-7szi: Cryo-EM structure of OmpK36-TraN mating pair stabilization protei... -

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Basic information

Entry
Database: PDB / ID: 7szi
TitleCryo-EM structure of OmpK36-TraN mating pair stabilization proteins from carbapenem-resistant Klebsiella pneumoniae
Components
  • OmpK36
  • TraN
KeywordsMEMBRANE PROTEIN / Bacterial Conjugation / Mating pair stabilization / Single Particle Analysis / Outer Membrane Protein Complex
Function / homology
Function and homology information


porin activity / pore complex / cell outer membrane / monoatomic ion transmembrane transport / metal ion binding
Similarity search - Function
Porin, gammaproteobacterial / Porin, Gram-negative type, conserved site / General diffusion Gram-negative porins signature. / Gram-negative porin / Porin, Gram-negative type / : / Porin domain superfamily
Similarity search - Domain/homology
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBeltran, L.C. / Seddon, C. / Beis, K. / Frankel, G. / Egelman, E.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5T32AI007046-45 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122510 United States
CitationJournal: 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
DepositionNov 27, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: OmpK36
B: OmpK36
C: OmpK36
D: TraN


Theoretical massNumber of molelcules
Total (without water)115,0844
Polymers115,0844
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein OmpK36


Mass: 38058.918 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Klebsiella pneumoniae (bacteria) / Strain: ICC8001 / References: UniProt: D6QLY0
#2: Protein/peptide TraN


Mass: 906.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: pKpQIL / Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Strain: ST 258 / Plasmid: pTAMAHISTEV
Details (production host): TraN gene (D28 to Q651)cells and expressed in Terrific Broth (TB)restriction enzyme sites. The construct was transformed into E. coli C43 (DE3) competent His7-tag and a ...Details (production host): TraN gene (D28 to Q651)cells and expressed in Terrific Broth (TB)restriction enzyme sites. The construct was transformed into E. coli C43 (DE3) competent His7-tag and a tobacco etch virus (TEV) cleavage site using the NcoI and XhoIfrom pKpQIL was subcloned into the pTAMAHISTEV vector with an N-
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Conjugal mating pair stabilizing complex OmpK36 and TraN
Type: COMPLEX
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.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Strain: ICC8001
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Plasmid: pTAMAHISTEV
Buffer solutionpH: 7.8 / Details: SEC Buffer
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium ChlorideNaCl1
210 mMHepesHepes1
30.03 percentDDMDDM1
SpecimenConc.: 0.033 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 4.78 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 13688
EM imaging opticsEnergyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
4cryoSPARC3.2.0CTF correction
9PHENIX1.19model refinement
12cryoSPARC3.2.0classification
13cryoSPARC3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13780567
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 598537 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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