+Open data
-Basic information
Entry | Database: PDB / ID: 7nsu | ||||||
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Title | ColicinE9 intact translocation complex | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / bacteriocin complex / import / membrane | ||||||
Function / homology | Function and homology information ABC-type vitamin B12 transporter activity / colicin transmembrane transporter activity / extrachromosomal circular DNA / cell cycle / cobalamin transport / protein import / porin activity / monoatomic ion channel complex / pore complex / protein homotrimerization ...ABC-type vitamin B12 transporter activity / colicin transmembrane transporter activity / extrachromosomal circular DNA / cell cycle / cobalamin transport / protein import / porin activity / monoatomic ion channel complex / pore complex / protein homotrimerization / transmembrane transporter complex / monoatomic ion channel activity / lipopolysaccharide binding / cell outer membrane / disordered domain specific binding / protein transport / monoatomic ion transmembrane transport / endonuclease activity / killing of cells of another organism / periplasmic space / Hydrolases; Acting on ester bonds / defense response to bacterium / protein domain specific binding / cell division / lipid binding / calcium ion binding / protein-containing complex / identical protein binding / membrane / metal ion binding Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å | ||||||
Authors | Webby, M.N. / Kleanthous, C. / Lukoyanova, N. / Housden, N.G. | ||||||
Funding support | 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7nsu.cif.gz | 756.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7nsu.ent.gz | 639.9 KB | Display | PDB format |
PDBx/mmJSON format | 7nsu.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7nsu_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7nsu_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 7nsu_validation.xml.gz | 77.1 KB | Display | |
Data in CIF | 7nsu_validation.cif.gz | 115.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ns/7nsu ftp://data.pdbj.org/pub/pdb/validation_reports/ns/7nsu | HTTPS FTP |
-Related structure data
Related structure data | 12577MC 7nstC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 37114.250 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (strain K12) (bacteria) Strain: K12 / Gene: ompF, cmlB, coa, cry, tolF, b0929, JW0912 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P02931 #2: Protein | | Mass: 61707.246 Da / Num. of mol.: 1 / Mutation: A33C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: col, cei / Production host: Escherichia coli (E. coli) References: UniProt: P09883, Hydrolases; Acting on ester bonds #3: Protein | | Mass: 46000.285 Da / Num. of mol.: 1 / Mutation: P201C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (strain K12) (bacteria) Strain: K12 / Gene: tolB, FAZ83_17845 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A6D2XIU5 #4: Protein | | Mass: 66386.180 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (strain K12) (bacteria) Strain: K12 / Gene: btuB, bfe, cer, dcrC, b3966, JW3938 / Production host: Escherichia coli (E. coli) / References: UniProt: P06129 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ColicinE9 intact translocon complex / Type: COMPLEX Details: Bacteriocin colicinE9 bound to outer membrane protein receptor BtuB and translocator ompF. Disulphide linked to tolB. Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.256 MDa |
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.9 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: PELCO easiGlow, Ted Pella Inc, USA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 92 % / Chamber temperature: 277.15 K 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 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 47619 X / Calibrated magnification: 47755 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1230 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Sampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 |
-Processing
Software |
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EM software |
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Image processing | Details: data recorded as gain corrected mrc files | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83697 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 198.91 Å2 | ||||||||||||||||||||||||
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