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- EMDB-21960: Colicin E1 fragment in nanodisc-embedded TolC -

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Basic information

Entry
Database: EMDB / ID: EMD-21960
TitleColicin E1 fragment in nanodisc-embedded TolC
Map dataC3-symmetrized map of nanodisc-embedded TolC
Sample
  • Complex: Outer membrane protein TolC
    • Protein or peptide: Outer membrane protein TolC
Keywordsantibiotic efflux / bacteriocin / TRANSPORT PROTEIN
Function / homology
Function and homology information


MacAB-TolC complex / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane / porin activity / bile acid and bile salt transport ...MacAB-TolC complex / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane / porin activity / bile acid and bile salt transport / monoatomic ion channel activity / efflux transmembrane transporter activity / cell outer membrane / response to toxic substance / outer membrane-bounded periplasmic space / monoatomic ion transmembrane transport / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane
Similarity search - Function
Type I secretion outer membrane protein, TolC / : / Outer membrane efflux protein / Outer membrane efflux protein
Similarity search - Domain/homology
Outer membrane protein TolC
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsKaelber JT / Budiardjo SJ
Funding support United States, 3 items
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, 2020-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.435
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.435
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6wxi
  • Surface level: 0.435
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21960.png

Downloads & links

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Map

FileDownload / File: emd_21960.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC3-symmetrized map of nanodisc-embedded TolC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 180 pix.
= 183.24 Å		1.02 Å/pix.
x 180 pix.
= 183.24 Å		1.02 Å/pix.
x 180 pix.
= 183.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.018 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.435 / Movie #1: 0.435
Minimum - Maximum-0.3198881 - 1.2916085
Average (Standard dev.)0.009030847 (±0.075726464)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 183.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0181.0181.018
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z183.240183.240183.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.3201.2920.009

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Supplemental data

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Mask #1

Fileemd_21960_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unmasked, unfiltered cryoSPARC half-map B of nanodisc-embedded TolC

Fileemd_21960_half_map_1.map
Annotationunmasked, unfiltered cryoSPARC half-map B of nanodisc-embedded TolC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unmasked, unfiltered cryoSPARC half-map A of nanodisc-embedded TolC

Fileemd_21960_half_map_2.map
Annotationunmasked, unfiltered cryoSPARC half-map A of nanodisc-embedded TolC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Outer membrane protein TolC

EntireName: Outer membrane protein TolC
Components
  • Complex: Outer membrane protein TolC
    • Protein or peptide: Outer membrane protein TolC

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Supramolecule #1: Outer membrane protein TolC

SupramoleculeName: Outer membrane protein TolC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 53.741 KDa

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Macromolecule #1: Outer membrane protein TolC

MacromoleculeName: Outer membrane protein TolC / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 53.783355 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLPILIG LSLSGFSSLS QAENLMQVYQ QARLSNPELR KSAADRDAAF EKINEARSPL LPQLGLGADY TYSNGYRDAN GINSNATSA SLQLTQSIFD MSKWRALTLQ EKAAGIQDVT YQTDQQTLIL NTATAYFNVL NAIDVLSYTQ AQKEAIYRQL D QTTQRFNV ...String:
MKKLLPILIG LSLSGFSSLS QAENLMQVYQ QARLSNPELR KSAADRDAAF EKINEARSPL LPQLGLGADY TYSNGYRDAN GINSNATSA SLQLTQSIFD MSKWRALTLQ EKAAGIQDVT YQTDQQTLIL NTATAYFNVL NAIDVLSYTQ AQKEAIYRQL D QTTQRFNV GLVAITDVQN ARAQYDTVLA NEVTARNNLD NAVEQLRQIT GNYYPELAAL NVENFKTDKP QPVNALLKEA EK RNLSLLQ ARLSQDLARE QIRQAQDGHL PTLDLTASTG ISDTSYSGSK TRGAAGTQYD DSNMGQNKVG LSFSLPIYQG GMV NSQVKQ AQYNFVGASE QLESAHRSVV QTVRSSFNNI NASISSINAY KQAVVSAQSS LDAMEAGYSV GTRTIVDVLD ATTT LYNAK QELANARYNY LINQLNIKSA LGTLNEQDLL ALNNALSKPV STNPENVAPQ TPEQNAIADG YAPDSPAPVV QQTSA RTTT SNGHNPFRN

UniProtKB: Outer membrane protein TolC

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
0.04 molarNaClsodium chloride
0.02 molarTris
GridModel: UltrAuFoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 16443 / Average exposure time: 8.0 sec. / Average electron dose: 33.04 e/Å2 / Details: 5 frames per second
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.593 µm / Calibrated defocus min: 0.47800000000000004 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2092678
Startup modelType of model: OTHER / Details: ab initio model, cryoSPARC 2
Final reconstructionNumber classes used: 17 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Software - details: non-uniform / Number images used: 196158
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.14.2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.14.2)
Final 3D classificationNumber classes: 64 / Software - Name: cryoSPARC (ver. 2.14.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6wxh


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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