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- PDB-7nsu: ColicinE9 intact translocation complex -

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Basic information

Entry
Database: PDB / ID: 7nsu
TitleColicinE9 intact translocation complex
Components
  • Colicin-E9
  • Outer membrane protein F
  • Tol-Pal system protein TolB
  • Vitamin B12 transporter BtuB
KeywordsMEMBRANE PROTEIN / bacteriocin complex / import / membrane
Function / homology
Function and homology information


ABC-type vitamin B12 transporter activity / colicin transmembrane transporter activity / extrachromosomal circular DNA / cobalamin transport / siderophore uptake transmembrane transporter activity / protein import / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization ...ABC-type vitamin B12 transporter activity / colicin transmembrane transporter activity / extrachromosomal circular DNA / cobalamin transport / siderophore uptake transmembrane transporter activity / protein import / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization / transmembrane transporter complex / 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 / calcium ion binding / protein-containing complex / identical protein binding / membrane / metal ion binding
Similarity search - Function
: / TonB-dependent vitamin B12 transporter BtuB / 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 ...: / TonB-dependent vitamin B12 transporter BtuB / 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 / TonB-dependent receptor (TBDR) proteins signature 1. / Cloacin colicin family / Colicin-like bacteriocin tRNase domain / Pyosin/cloacin translocation domain / Pyosin/cloacin translocation domain superfamily / TonB box, conserved site / TonB-dependent receptor, conserved site / TonB-dependent receptor (TBDR) proteins signature 2. / 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 / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent receptor-like, beta-barrel domain superfamily / TonB-dependent Receptor Plug Domain / 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 / Vitamin B12 transporter BtuB / Colicin-E9
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsWebby, M.N. / Kleanthous, C. / Lukoyanova, N. / Housden, N.G.
Funding support1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Nov 10, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Nov 17, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

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  • EMDB-12577
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Assembly

Deposited unit
A: Outer membrane protein F
B: Outer membrane protein F
C: Outer membrane protein F
D: Colicin-E9
E: Tol-Pal system protein TolB
F: Vitamin B12 transporter BtuB


Theoretical massNumber of molelcules
Total (without water)285,4366
Polymers285,4366
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15960 Å2
ΔGint-74 kcal/mol
Surface area102620 Å2
MethodPISA

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Components

#1: Protein Outer membrane protein F / Outer membrane protein 1A / Outer membrane protein B / Outer membrane protein IA / Porin OmpF


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 Colicin-E9


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 Tol-Pal system protein TolB


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 Vitamin B12 transporter BtuB / Cobalamin receptor / Outer membrane cobalamin translocator


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

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
Molecular weightValue: 0.256 MDa
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.9
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: PELCO easiGlow, Ted Pella Inc, USA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameCategory
2EPUimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
Image processingDetails: data recorded as gain corrected mrc files
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83697 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 198.91 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003318960
ELECTRON MICROSCOPYf_angle_d0.69925744
ELECTRON MICROSCOPYf_chiral_restr0.04592697
ELECTRON MICROSCOPYf_plane_restr0.0053458
ELECTRON MICROSCOPYf_dihedral_angle_d14.5136748

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