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- PDB-7ng9: Trimeric efflux pump Klebsiella TolC -

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Basic information

Entry
Database: PDB / ID: 7ng9
TitleTrimeric efflux pump Klebsiella TolC
ComponentsOuter membrane channel protein
KeywordsMEMBRANE PROTEIN / Efflux pump / trimer
Function / homologyType I secretion outer membrane protein, TolC / Outer membrane efflux protein / Outer membrane efflux protein / efflux pump complex / porin activity / efflux transmembrane transporter activity / cell outer membrane / Outer membrane channel protein TolC
Function and homology information
Biological speciesKlebsiella quasipneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWebby, M.N. / Housden, N.G. / Kleanthous, C.
Funding support1items
OrganizationGrant numberCountry
Wellcome Trust
CitationJournal: Nat Commun / Year: 2021
Title: Toxin import through the antibiotic efflux channel TolC.
Authors: Nicholas G Housden / Melissa N Webby / Edward D Lowe / Tarick J El-Baba / Renata Kaminska / Christina Redfield / Carol V Robinson / Colin Kleanthous /
Abstract: Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a ...Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.
History
DepositionFeb 8, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / em_admin / em_map / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _em_map.file
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Assembly

Deposited unit
A: Outer membrane channel protein
B: Outer membrane channel protein
C: Outer membrane channel protein


Theoretical massNumber of molelcules
Total (without water)162,1763
Polymers162,1763
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14540 Å2
ΔGint-59 kcal/mol
Surface area55650 Å2
MethodPISA

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Components

#1: Protein Outer membrane channel protein / Outer membrane channel protein TolC


Mass: 54058.676 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella quasipneumoniae (bacteria)
Gene: tolC, CP911_05930, DBL00_17640, G5630_14450, SAMEA104168726_00630, SAMEA3531848_02756
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1C3Q001

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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: Homotrimer of TolC from klebsiella / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Klebsiella quasipneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.9
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameCategory
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 188171 / Symmetry type: POINT
Atomic model buildingPDB-ID: 1EK9

1ek9


Accession code: 1EK9 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 39.96 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00659870
ELECTRON MICROSCOPYf_angle_d0.472913401
ELECTRON MICROSCOPYf_chiral_restr0.03561566
ELECTRON MICROSCOPYf_plane_restr0.00251785
ELECTRON MICROSCOPYf_dihedral_angle_d17.42823636

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