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- EMDB-2372: Negative stain EM structure of the trypsin digested Colicin E9 tr... -

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Basic information

Entry
Database: EMDB / ID: EMD-2372
TitleNegative stain EM structure of the trypsin digested Colicin E9 translocon complex
Map dataNegative stain EM structure of the trypsin digested Colicin E9 translocon complex comprising OmpF trimer and TolB connected by colicin fragment (residues 2-122).
Sample
  • Sample: Trypsin digested colicin E9 translocon complex comprising OmpF trimer and TolB connected by Colicin fragment (residues 2-122).
  • Protein or peptide: Outer membrane porin 1a (Ia;b;F)
  • Protein or peptide: Colicin-E9
  • Protein or peptide: Protein TolB
KeywordsColicins / Porins / Translocon
Function / homology
Function and homology information


cellular response to bacteriocin / regulation of membrane invagination / cell septum assembly / bacteriocin transport / extrachromosomal circular DNA / protein import / cell division site / protein transport / outer membrane-bounded periplasmic space / endonuclease activity ...cellular response to bacteriocin / regulation of membrane invagination / cell septum assembly / bacteriocin transport / extrachromosomal circular DNA / protein import / cell division site / protein transport / outer membrane-bounded periplasmic space / endonuclease activity / killing of cells of another organism / Hydrolases; Acting on ester bonds / periplasmic space / defense response to bacterium / protein domain specific binding / cell division / protein-containing complex binding / protein-containing complex / 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, DNase domain superfamily / Colicin/Pyocin-S2, DNase domain / Colicin, receptor domain / Coiled-coil receptor-binding R-domain of colicin E2 / Cloacin colicin family ...TolB, N-terminal / Tol-Pal system protein TolB / TolB amino-terminal domain / WD40-like beta propeller / WD40-like Beta Propeller Repeat / Colicin/pyocin, DNase domain superfamily / Colicin/Pyocin-S2, DNase domain / 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 / His-Me finger superfamily / Six-bladed beta-propeller, TolB-like
Similarity search - Domain/homology
: / Colicin-E9 / Tol-Pal system protein TolB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsLukoyanova N / Housden NG / Hopper JTS / Rodriguez-Larrea D / Wojdyla JA / Klein A / Kaminska R / Bayley H / Robinson CV / Kleanthous C / Saibil HR
CitationJournal: Science / Year: 2013
Title: Intrinsically disordered protein threads through the bacterial outer-membrane porin OmpF.
Authors: Nicholas G Housden / Jonathan T S Hopper / Natalya Lukoyanova / David Rodriguez-Larrea / Justyna A Wojdyla / Alexander Klein / Renata Kaminska / Hagan Bayley / Helen R Saibil / Carol V ...Authors: Nicholas G Housden / Jonathan T S Hopper / Natalya Lukoyanova / David Rodriguez-Larrea / Justyna A Wojdyla / Alexander Klein / Renata Kaminska / Hagan Bayley / Helen R Saibil / Carol V Robinson / Colin Kleanthous /
Abstract: Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) ...Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9's unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus, an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death.
History
DepositionMay 10, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseJul 17, 2013-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0123
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0123
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2372_ima...

Downloads & links

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Map

FileDownload / File: emd_2372.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM structure of the trypsin digested Colicin E9 translocon complex comprising OmpF trimer and TolB connected by colicin fragment (residues 2-122).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 256 pix.
= 460.8 Å		1.8 Å/pix.
x 256 pix.
= 460.8 Å		1.8 Å/pix.
x 256 pix.
= 460.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0123 / Movie #1: 0.0123
Minimum - Maximum-0.00128456 - 0.07485089
Average (Standard dev.)0.00008634 (±0.00161523)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 460.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0010.0750.000

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Supplemental data

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Sample components

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Entire : Trypsin digested colicin E9 translocon complex comprising OmpF tr...

EntireName: Trypsin digested colicin E9 translocon complex comprising OmpF trimer and TolB connected by Colicin fragment (residues 2-122).
Components
  • Sample: Trypsin digested colicin E9 translocon complex comprising OmpF trimer and TolB connected by Colicin fragment (residues 2-122).
  • Protein or peptide: Outer membrane porin 1a (Ia;b;F)
  • Protein or peptide: Colicin-E9
  • Protein or peptide: Protein TolB

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Supramolecule #1000: Trypsin digested colicin E9 translocon complex comprising OmpF tr...

SupramoleculeName: Trypsin digested colicin E9 translocon complex comprising OmpF trimer and TolB connected by Colicin fragment (residues 2-122).
type: sample / ID: 1000
Oligomeric state: OmpF trimer and TolB connected by colicin fragment
Number unique components: 3
Molecular weightExperimental: 166.528 KDa / Theoretical: 166.572 KDa / Method: native state ESI-MS

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Macromolecule #1: Outer membrane porin 1a (Ia;b;F)

MacromoleculeName: Outer membrane porin 1a (Ia;b;F) / type: protein_or_peptide / ID: 1 / Name.synonym: OmpF / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli / Location in cell: outer membrane
Molecular weightExperimental: 111.294 KDa / Theoretical: 111.253 KDa
SequenceUniProtKB: UNIPROTKB: C4ZQ55

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Macromolecule #2: Colicin-E9

MacromoleculeName: Colicin-E9 / type: protein_or_peptide / ID: 2 / Name.synonym: A33C colicin E9 residues 2-122 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli / Location in cell: outer membrane
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Colicin-E9

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Macromolecule #3: Protein TolB

MacromoleculeName: Protein TolB / type: protein_or_peptide / ID: 3 / Name.synonym: P201C TolBHis6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli / Location in cell: periplasm
Molecular weightTheoretical: 44 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Tol-Pal system protein TolB

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 6.5 / Details: 1% (w/v) n-octyl-D-glucopyranoside, 20 mM MES
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 60 seconds
GridDetails: 300 square mesh copper grid with ~9nm carbon support, negatively glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification
DateDec 11, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Digitization - Sampling interval: 15 µm / Number real images: 92 / Average electron dose: 25 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80925 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsIndividual images (n=15,500) of the digested translocon complex were interactively selected from micrographs using the EMAN/Boxer software package. Particle images were normalized and band-pass filtered between 150 A and 8 A. Both angular reconstitution sub-tomogram averages were used as initial models for independent projection matching with SPIDER. Independently of initial model for projection matching, final reconstructions displayed similar features at ~20 A resolution estimated by FSC at 0.5 correlation.
CTF correctionDetails: phases corrected only
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF
Software - Name: CTFFIND, Spider, EMAN1/Boxer, Imagic, IMOD, Peet
Details: Both reconstruction by angular reconstitution and selected sub-tomogram averages were used as initial models for independent projection matching with SPIDER. Independently of initial model ...Details: Both reconstruction by angular reconstitution and selected sub-tomogram averages were used as initial models for independent projection matching with SPIDER. Independently of initial model for projection matching, final reconstructions displayed similar features at ~20 A resolution estimated by FSC at 0.5 correlation
Number images used: 7000

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Atomic model buiding 1

Initial modelPDB ID:

4jml

SoftwareName: Chimera
Detailscrystal structures were manually fitted into the EM map
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3o0e

SoftwareName: Chimera
Detailscrystal structures were manually fitted into the EM map
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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