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- EMDB-25567: Cryo-EM structure of OmpK36-TraN mating pair stabilization protei... -

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Basic information

Entry
Database: EMDB / ID: EMD-25567
TitleCryo-EM structure of OmpK36-TraN mating pair stabilization proteins from carbapenem-resistant Klebsiella pneumoniae
Map dataWhole OmpK36-TraN map
Sample
  • Complex: Conjugal mating pair stabilizing complex OmpK36 and TraN
    • Protein or peptide: OmpK36
    • Protein or peptide: TraN
Function / homology
Function and homology information


porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBeltran LC / Seddon C / Beis K / Frankel G / Egelman EH
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25567.png

Downloads & links

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Map

FileDownload / File: emd_25567.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWhole OmpK36-TraN map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.138
Minimum - Maximum-0.40753052 - 0.9880569
Average (Standard dev.)0.00037987935 (±0.0251262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: TraN loop only

Fileemd_25567_additional_1.map
AnnotationTraN loop only
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: OmpK36 Barrels only

Fileemd_25567_additional_2.map
AnnotationOmpK36 Barrels only
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: TraN Flexible Tail

Fileemd_25567_additional_3.map
AnnotationTraN Flexible Tail
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Conjugal mating pair stabilizing complex OmpK36 and TraN

EntireName: Conjugal mating pair stabilizing complex OmpK36 and TraN
Components
  • Complex: Conjugal mating pair stabilizing complex OmpK36 and TraN
    • Protein or peptide: OmpK36
    • Protein or peptide: TraN

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Supramolecule #1: Conjugal mating pair stabilizing complex OmpK36 and TraN

SupramoleculeName: 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.
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Strain: ICC8001
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pTAMAHISTEV
Molecular weightExperimental: 120 KDa

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Macromolecule #1: OmpK36

MacromoleculeName: OmpK36 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Strain: ICC8001
Molecular weightTheoretical: 38.058918 KDa
SequenceString: 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

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Macromolecule #2: TraN

MacromoleculeName: TraN / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Strain: ST 258
Molecular weightTheoretical: 906.964 Da
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
CSGGQNTHC

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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

Concentration0.033 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chlorideSodium Chloride
10.0 mMHepesHepes
0.03 percentDDMDDM

Details: SEC Buffer
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 opticsEnergy filter - Slit width: 10 eV
Image recordingFilm 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

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Image processing

Particle selectionNumber selected: 13780567
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2.0)
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied 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

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7szi:
Cryo-EM structure of OmpK36-TraN mating pair stabilization proteins from carbapenem-resistant Klebsiella pneumoniae

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