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- EMDB-12309: Trimeric efflux pump Klebsiella TolC in complex with KlebC -

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Basic information

Entry
Database: EMDB / ID: EMD-12309
TitleTrimeric efflux pump Klebsiella TolC in complex with KlebC
Map dataLocScale sharpened map
Sample
  • Complex: Homotrimer of TolC from klebsiella in complex with KlebC
    • Protein or peptide: Outer membrane channel protein
    • Protein or peptide: Klebicin C activity
KeywordsEfflux pump / trimer / complex / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWebby MN / Housden NG
Funding support1 items
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, 2021-
Header (metadata) releaseJun 30, 2021-
Map releaseJun 30, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.145
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.145
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ng8
  • Surface level: 0.145
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ng8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12309.png

Downloads & links

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Map

FileDownload / File: emd_12309.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocScale sharpened map
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.145 / Movie #1: 0.145
Minimum - Maximum-0.26812598 - 0.667354
Average (Standard dev.)0.0017847172 (±0.019918647)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z246.600246.600246.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2680.6670.002

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Supplemental data

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Additional map: Sharpened map from relion locres job. Requires Zflip

Fileemd_12309_additional_1.map
AnnotationSharpened map from relion locres job. Requires Zflip
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map from relion refine3D job. Requires zflip

Fileemd_12309_additional_2.map
AnnotationUnsharpened map from relion refine3D job. Requires zflip
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocess masked map from relion. Requires Zflip

Fileemd_12309_additional_3.map
AnnotationPostprocess masked map from relion. Requires Zflip
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homotrimer of TolC from klebsiella in complex with KlebC

EntireName: Homotrimer of TolC from klebsiella in complex with KlebC
Components
  • Complex: Homotrimer of TolC from klebsiella in complex with KlebC
    • Protein or peptide: Outer membrane channel protein
    • Protein or peptide: Klebicin C activity

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Supramolecule #1: Homotrimer of TolC from klebsiella in complex with KlebC

SupramoleculeName: Homotrimer of TolC from klebsiella in complex with KlebC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Klebsiella quasipneumoniae (bacteria)

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Macromolecule #1: Outer membrane channel protein

MacromoleculeName: Outer membrane channel protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella quasipneumoniae (bacteria)
Molecular weightTheoretical: 54.058676 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLPILIG LSLTGFSAMS QAENLLQVYQ QARISNPDLR KSAADRDAAF EKINEARSPL LPQLGLGADY TYTSGFRDYK DQNSNVTSG SLQLTQVLFD MSKWRALTLQ EKAAGIQDVT YQTDQQTLIL NTATAYFKVL AAIDTLSYTE AQKQAIYRQL D QTTQRFNV ...String:
MKKLLPILIG LSLTGFSAMS QAENLLQVYQ QARISNPDLR KSAADRDAAF EKINEARSPL LPQLGLGADY TYTSGFRDYK DQNSNVTSG SLQLTQVLFD MSKWRALTLQ EKAAGIQDVT YQTDQQTLIL NTATAYFKVL AAIDTLSYTE AQKQAIYRQL D QTTQRFNV GLVAITDVQN ARSQYDAVLA NEVTARNDLD NAVEELRQVT GNYYPELASL NVDGFKTSKP QAVNALLKEA EN RNLSLLQ ARLNQDLARE QIRQAQDGHL PTLDLNASSG VSNNRYSGSK SISQDADIGQ NKIGLSFSLP LYQGGMVNSQ VKQ AQYNFV GASEQLESAH RSVVQTVRSS FNNVNASISS INAYKQAVVS AQSSLDAMEA GYSVGTRTIV DVLDATTTLY NAKQ QLSNA RYNYLINELN IKSALGTLNE QDLIALNNTL GKPISTSADS VAPENPQQDA TADGYGNTTA AMKPASARTT THSSG SNPF RQLEHHHHHH

UniProtKB: Outer membrane channel protein TolC

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Macromolecule #2: Klebicin C activity

MacromoleculeName: Klebicin C activity / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella quasipneumoniae (bacteria)
Molecular weightTheoretical: 28.629162 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MADNQPVPLT PAPPGMVSLG VNENGEEEMT VIGGDGSGTG FSGNEAPIIP GSGSLQADLG KKSLTRLQAE SSAAIHATAK WTTENLAKT QAAQAERAKA AMLSQQAAKA KQAKLTQHLK DVVDRALQNN KTRPTVIDLA HQNNQQMAAM AEFIGRQKAI E EARKKAER ...String:
MADNQPVPLT PAPPGMVSLG VNENGEEEMT VIGGDGSGTG FSGNEAPIIP GSGSLQADLG KKSLTRLQAE SSAAIHATAK WTTENLAKT QAAQAERAKA AMLSQQAAKA KQAKLTQHLK DVVDRALQNN KTRPTVIDLA HQNNQQMAAM AEFIGRQKAI E EARKKAER EAKRAEEAYQ AALRAQEEEQ RKQAEIERKL QEARKQEAAA KAKAEADRIA AEKAEAEARA KAEAERRKAE EA RKALFAK AGIKDTPGCL EHHHHHH

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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

Concentration0.8 mg/mL
BufferpH: 7.9
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 435603
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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