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- EMDB-12576: ColicinE9 partial translocation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-12576
TitleColicinE9 partial translocation complex
Map dataOutput unsharpened map from relion refine3D job, sharpened using deepEMhancer tight target model
Sample
  • Complex: ColicinE9 intact translocon complex
    • Protein or peptide: Outer membrane protein F
    • Protein or peptide: Colicin-E9
    • Protein or peptide: Tol-Pal system protein TolB
Keywordsbacteriocin complex / import-stalled / MEMBRANE PROTEIN
Function / homology
Function and homology information


colicin transmembrane transporter activity / extrachromosomal circular DNA / protein import / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization / monoatomic ion transmembrane transport / lipopolysaccharide binding / cell outer membrane ...colicin transmembrane transporter activity / extrachromosomal circular DNA / protein import / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization / monoatomic ion transmembrane transport / lipopolysaccharide binding / cell outer membrane / disordered domain specific binding / protein transport / monoatomic ion channel activity / endonuclease activity / killing of cells of another organism / periplasmic space / Hydrolases; Acting on ester bonds / defense response to bacterium / cell cycle / protein domain specific binding / cell division / lipid binding / protein-containing complex / identical protein binding / membrane / metal ion binding
Similarity search - Function
: / TolB, N-terminal / Tol-Pal system protein TolB / TolB amino-terminal domain / WD40-like beta propeller / WD40-like Beta Propeller Repeat / Colicin/Pyocin-S2, DNase domain / Colicin/pyocin, DNase domain superfamily / Colicin, receptor domain / Coiled-coil receptor-binding R-domain of colicin E2 ...: / TolB, N-terminal / Tol-Pal system protein TolB / TolB amino-terminal domain / WD40-like beta propeller / WD40-like Beta Propeller Repeat / Colicin/Pyocin-S2, DNase domain / Colicin/pyocin, DNase domain superfamily / Colicin, receptor domain / Coiled-coil receptor-binding R-domain of colicin E2 / Cloacin colicin family / Colicin-like bacteriocin tRNase domain / Pyosin/cloacin translocation domain / Pyosin/cloacin translocation domain superfamily / Porin, gammaproteobacterial / Porin, Gram-negative type, conserved site / General diffusion Gram-negative porins signature. / Porin domain, Gram-negative type / Gram-negative porin / Porin, Gram-negative type / His-Me finger superfamily / Porin domain superfamily / Six-bladed beta-propeller, TolB-like
Similarity search - Domain/homology
Tol-Pal system protein TolB / Outer membrane porin F / Colicin-E9
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWebby MN / Kleanthous C
Funding support1 items
OrganizationGrant numberCountry
European Research Council (ERC)
CitationJournal: EMBO J / Year: 2021
Title: Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.
Authors: Marie-Louise R Francis / Melissa N Webby / Nicholas G Housden / Renata Kaminska / Emma Elliston / Boonyaporn Chinthammit / Natalya Lukoyanova / Colin Kleanthous /
Abstract: Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these ...Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.
History
DepositionMar 8, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 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: 0.0391
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0391
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nst
  • Surface level: 0.0391
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12576.png

Downloads & links

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Map

FileDownload / File: emd_12576.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOutput unsharpened map from relion refine3D job, sharpened using deepEMhancer tight target model
Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.0391 / Movie #1: 0.0391
Minimum - Maximum-0.0017193394 - 1.6808244
Average (Standard dev.)0.0010476976 (±0.023985384)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 293.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z293.160293.160293.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0021.6810.001

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Supplemental data

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Additional map: Unsharpened map from relion refine3D job

Fileemd_12576_additional_1.map
AnnotationUnsharpened map from relion refine3D job
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A from refine3D in relion3.1

Fileemd_12576_half_map_1.map
Annotationhalf map A from refine3D in relion3.1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B from refine3D in relion3.1

Fileemd_12576_half_map_2.map
Annotationhalf map B from refine3D in relion3.1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ColicinE9 intact translocon complex

EntireName: ColicinE9 intact translocon complex
Components
  • Complex: ColicinE9 intact translocon complex
    • Protein or peptide: Outer membrane protein F
    • Protein or peptide: Colicin-E9
    • Protein or peptide: Tol-Pal system protein TolB

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Supramolecule #1: ColicinE9 intact translocon complex

SupramoleculeName: ColicinE9 intact translocon complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Bacteriocin colicinE9 bound to outer membrane protein receptor BtuB and translocator ompF. Disulphide linked to tolB.
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 256 KDa

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Macromolecule #1: Outer membrane protein F

MacromoleculeName: Outer membrane protein F / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 37.11425 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AEIYNKDGNK VDLYGKAVGL HYFSKGNGEN SYGGNGDMTY ARLGFKGETQ INSDLTGYGQ WEYNFQGNNS EGADAQTGNK TRLAFAGLK YADVGSFDYG RNYGVVYDAL GYTDMLPEFG GDTAYSDDFF VGRVGGVATY RNSNFFGLVD GLNFAVQYLG K NERDTARR ...String:
AEIYNKDGNK VDLYGKAVGL HYFSKGNGEN SYGGNGDMTY ARLGFKGETQ INSDLTGYGQ WEYNFQGNNS EGADAQTGNK TRLAFAGLK YADVGSFDYG RNYGVVYDAL GYTDMLPEFG GDTAYSDDFF VGRVGGVATY RNSNFFGLVD GLNFAVQYLG K NERDTARR SNGDGVGGSI SYEYEGFGIV GAYGAADRTN LQEAQPLGNG KKAEQWATGL KYDANNIYLA ANYGETRNAT PI TNKFTNT SGFANKTQDV LLVAQYQFDF GLRPSIAYTK SKAKDVEGIG DVDLVNYFEV GATYYFNKNM STYVDYIINQ IDS DNKLGV GSDDTVAVGI VYQF

UniProtKB: Outer membrane porin F

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Macromolecule #2: Colicin-E9

MacromoleculeName: Colicin-E9 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 31.563652 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSGGDGRGHN TGAHSTSGNI NGGPTGIGVS GGCSDGSGWS SENNPWGGGS GSGIHWGGGS GRGNGGGNGN SGGGSGTGGN LSAVAAPVA FGFPALSTPG AGGLAVSISA SELSAAIAGI IAKLKKVNLK FTPFGVVLSS LIPSEIAKDD PNMMSKIVTS L PADDITES ...String:
MSGGDGRGHN TGAHSTSGNI NGGPTGIGVS GGCSDGSGWS SENNPWGGGS GSGIHWGGGS GRGNGGGNGN SGGGSGTGGN LSAVAAPVA FGFPALSTPG AGGLAVSISA SELSAAIAGI IAKLKKVNLK FTPFGVVLSS LIPSEIAKDD PNMMSKIVTS L PADDITES PVSSLPLDKA TVNVNVRVVD DVKDERQNIS VVSGVPMSVP VVDAKPTERP GVFTASIPGA PVLNISVNDS TP AVQTLSP GVTNNTDKDV RPAGFTQGGN TRDAVIRFPK DSGHNAVYVS VSDVLSPDQV KQRQDEENRR QQEWDAT

UniProtKB: Colicin-E9

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Macromolecule #3: Tol-Pal system protein TolB

MacromoleculeName: Tol-Pal system protein TolB / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 46.000285 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKQALRVAFG FLILWASVLH AEVRIVIDSG VDSGRPIGVV PFQWAGPGAA PEDIGGIVAA DLRNSGKFNP LDRARLPQQP GSAQEVQPA AWSALGIDAV VVGQVTPNPD GSYNVAYQLV DTGGAPGTVL AQNSYKVNKQ WLRYAGHTAS DEVFEKLTGI K GAFRTRIA ...String:
MKQALRVAFG FLILWASVLH AEVRIVIDSG VDSGRPIGVV PFQWAGPGAA PEDIGGIVAA DLRNSGKFNP LDRARLPQQP GSAQEVQPA AWSALGIDAV VVGQVTPNPD GSYNVAYQLV DTGGAPGTVL AQNSYKVNKQ WLRYAGHTAS DEVFEKLTGI K GAFRTRIA YVVQTNGGQF PYELRVSDYD GYNQFVVHRS PQCLMSPAWS PDGSKLAYVT FESGRSALVI QTLANGAVRQ VA SFPRHNG APAFSPDGSK LAFALSKTGS LNLYVMDLAS GQIRQVTDGR SNNTEPTWFP DSQNLAFTSD QAGRPQVYKV NIN GGAPQR ITWEGSQNQD ADVSSDGKFM VMVSSNGGQQ HIAKQDLATG GVQVLSSTFL DETPSLAPNG TMVIYSSSQG MGSV LNLVS TDGRFKARLP ATDGQVKFPA WSPYL

UniProtKB: Tol-Pal system protein TolB

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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

Concentration0.5 mg/mL
BufferpH: 7.9
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: GRAPHENE OXIDE / Support film - #0 - topology: CONTINUOUS / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: PELCO easiGlow, Ted Pella Inc, USA
VitrificationCryogen name: ETHANE / Chamber humidity: 92 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 3 uL of diluted translocon preparation were applied on freshly glow discharged grids coated with graphene oxide (as described in https://doi.org/10.1038/s41594-019-0355-2); after 30sec ...Details: 3 uL of diluted translocon preparation were applied on freshly glow discharged grids coated with graphene oxide (as described in https://doi.org/10.1038/s41594-019-0355-2); after 30sec waiting grids were blotted for 8-10 using -10 force and plunge frozen in liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / 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: 2 / Number real images: 3000 / Average exposure time: 10.0 sec. / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.23 µm / Calibrated magnification: 47755 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 47619
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailsdata recorded as gain corrected mrc files
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71190
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: RELION
FSC plot (resolution estimation)

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