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- PDB-6wxh: Colicin E1 fragment in nanodisc-embedded TolC -

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Basic information

Entry
Database: PDB / ID: 6wxh
TitleColicin E1 fragment in nanodisc-embedded TolC
Components
  • Colicin-E1
  • Outer membrane protein TolC
KeywordsANTIMICROBIAL PROTEIN / antibiotic efflux / bacteriocin / TRANSPORT PROTEIN
Function / homology
Function and homology information


negative regulation of ion transmembrane transporter activity / MacAB-TolC complex / pore-forming activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane ...negative regulation of ion transmembrane transporter activity / MacAB-TolC complex / pore-forming activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane / bile acid and bile salt transport / porin activity / efflux transmembrane transporter activity / monoatomic ion channel activity / cell outer membrane / response to organic cyclic compound / response to toxic substance / outer membrane-bounded periplasmic space / monoatomic ion transmembrane transport / defense response to Gram-negative bacterium / transmembrane transporter binding / killing of cells of another organism / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
Channel forming colicin, C-terminal cytotoxic / Channel forming colicin, C-terminal domain superfamily / Colicin pore forming domain / Channel forming colicins signature. / Type I secretion outer membrane protein, TolC / : / Outer membrane efflux protein / Outer membrane efflux protein
Similarity search - Domain/homology
Outer membrane protein TolC / Colicin-E1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsKaelber, J.T. / Budiardjo, S.J. / Firlar, E. / Case, D.A. / Ikujuni, A.P. / Slusky, J.S.G.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM128201 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM113117 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103638 United States
CitationJournal: Elife / Year: 2022
Title: Colicin E1 opens its hinge to plug TolC.
Authors: S Jimmy Budiardjo / Jacqueline J Stevens / Anna L Calkins / Ayotunde P Ikujuni / Virangika K Wimalasena / Emre Firlar / David A Case / Julie S Biteen / Jason T Kaelber / Joanna S G Slusky /
Abstract: The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed ...The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, that hijacks the outer membrane proteins (OMPs) TolC and BtuB to enter the cell. Here, we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryo-electron microscopy to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump, and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other OMP functions.
History
DepositionMay 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 12, 2021Provider: repository / Type: Initial release
Revision 1.1May 4, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 15, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-21959
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Outer membrane protein TolC
B: Outer membrane protein TolC
C: Outer membrane protein TolC
D: Colicin-E1


Theoretical massNumber of molelcules
Total (without water)182,8424
Polymers182,8424
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20090 Å2
ΔGint-68 kcal/mol
Surface area61930 Å2

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Components

#1: Protein Outer membrane protein TolC / Multidrug efflux pump subunit TolC / Outer membrane factor TolC


Mass: 53783.355 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12
Gene: tolC, colE1-i, mtcB, mukA, refI, toc, weeA, b3035, JW5503
Plasmid: pTrc99a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P02930
#2: Protein Colicin-E1


Mass: 21491.807 Da / Num. of mol.: 1 / Fragment: colE1-T residues 1-190
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cea / Plasmid: pET303 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P02978

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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

Component
IDNameTypeEntity IDParent-IDSource
1Colicin E1 fragment colE1-T inside nanodisc-embedded TolCCOMPLEXall0RECOMBINANT
2Outer membrane protein TolCCOMPLEX#11RECOMBINANT
3Colicin E1 fragment colE1-TCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.053741 MDaNO
210.02144974 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Escherichia coli BL21(DE3) (bacteria)469008pTrc99a
23Escherichia coli BL21(DE3) (bacteria)469008pET303
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1.04 Msodium chlorideNaCl1
2.02 MTris1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 205000 X / Calibrated defocus min: 400 nm / Calibrated defocus max: 2100 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 35.96 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 10644 / Details: 5 frames per second
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40

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Processing

SoftwareName: UCSF ChimeraX / Version: 0.92/v8 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Linux / Type: package
EM software
IDNameVersionCategoryDetails
2SerialEM3.7image acquisitionLD-Group
4CTFFIND4CTF correction
7Coot0.8.9.2-premodel fitting
8ISOLDE1.0b4.dev1model fitting
10cryoSPARC2.14.2initial Euler assignment
11cryoSPARC2.14.2final Euler assignment
12cryoSPARC2.14.2classification
13cryoSPARC2.14.23D reconstructionnon-uniform
20PHENIX1.14-3260model refinement
21Coot0.8.9.2-premodel refinement
22ISOLDE1.0b4.dev1model refinement
23Amber20model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 339779
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179834 / Algorithm: FOURIER SPACE
Details: Additional map (C3 refinement) is 2.81Angstrom resolution
Num. of class averages: 29 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: colE1 built ab initio TolC from 1TQQ
Atomic model buildingPDB-ID: 1TQQ

1tqq


Accession code: 1TQQ / Source name: PDB / Type: experimental model

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