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- EMDB-2299: Negative stain EM structure of the PTC3 holotoxin complex (TcdA1,... -

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Basic information

Entry
Database: EMDB / ID: EMD-2299
TitleNegative stain EM structure of the PTC3 holotoxin complex (TcdA1, TcdB2, TccC3) in prepore state
Map dataReconstruction of PTC3 holotoxin complex in prepore state
Sample
  • Sample: PTC3 (TcdA1, TcdB2, TccC3)
  • Protein or peptide: TcdA1
  • Protein or peptide: TccC3
  • Protein or peptide: TcdB2
KeywordsPhotorabdus / translocation / pore formation / bacterial toxin / ABC toxin / helical toxin
Function / homology
Function and homology information


extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / : / TcdA1, receptor binding domain ...Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / : / TcdA1, receptor binding domain / : / TcdA1, receptor binding domain / ABC toxin, N-terminal domain / ABC toxin N-terminal region / TcA receptor binding domain / TcA receptor binding domain / Insecticidal toxin complex/plasmid virulence protein / Tc toxin complex TcA, C-terminal TcB-binding domain / Neuraminidase-like domain / Salmonella virulence plasmid 28.1kDa A protein / Tc toxin complex TcA C-terminal TcB-binding domain / Neuraminidase-like domain / Rhs repeat-associated core / : / Integrin alpha, N-terminal
Similarity search - Domain/homology
Insecticidal toxin complex protein TcdB2 / Insecticidal toxin complex protein TccC3 / TcdA1
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 24.6 Å
AuthorsGatsogiannis C / Lang A E / Meusch D / Pfaumann V / Hofnagel O / Benz R / Aktories K / Raunser S
CitationJournal: Nature / Year: 2013
Title: A syringe-like injection mechanism in Photorhabdus luminescens toxins.
Authors: Christos Gatsogiannis / Alexander E Lang / Dominic Meusch / Vanda Pfaumann / Oliver Hofnagel / Roland Benz / Klaus Aktories / Stefan Raunser /
Abstract: Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills ...Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.
History
DepositionJan 25, 2013-
Header (metadata) releaseFeb 13, 2013-
Map releaseFeb 13, 2013-
UpdateApr 3, 2013-
Current statusApr 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2299.png

Downloads & links

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Map

FileDownload / File: emd_2299.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PTC3 holotoxin complex in prepore state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.64 Å/pix.
x 112 pix.
= 519.68 Å		4.64 Å/pix.
x 112 pix.
= 519.68 Å		4.64 Å/pix.
x 112 pix.
= 519.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.05874737 - 0.09712731
Average (Standard dev.)0.00082589 (±0.00667715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions112112112
Spacing112112112
CellA=B=C: 519.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.644.644.64
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z519.680519.680519.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-0.0590.0970.001

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Supplemental data

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Sample components

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Entire : PTC3 (TcdA1, TcdB2, TccC3)

EntireName: PTC3 (TcdA1, TcdB2, TccC3)
Components
  • Sample: PTC3 (TcdA1, TcdB2, TccC3)
  • Protein or peptide: TcdA1
  • Protein or peptide: TccC3
  • Protein or peptide: TcdB2

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Supramolecule #1000: PTC3 (TcdA1, TcdB2, TccC3)

SupramoleculeName: PTC3 (TcdA1, TcdB2, TccC3) / type: sample / ID: 1000
Oligomeric state: TcdB2/TccC3 bind to one homopentamer of TcdA1
Number unique components: 3
Molecular weightExperimental: 1.68 MDa / Theoretical: 1.68 MDa

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Macromolecule #1: TcdA1

MacromoleculeName: TcdA1 / type: protein_or_peptide / ID: 1 / Name.synonym: Toxin A / Number of copies: 5 / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightExperimental: 283 KDa / Theoretical: 283 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: CodonPlus
Recombinant plasmid: pCR-BluntII-TOPO, subcloned into pET-28a(+)
SequenceUniProtKB: TcdA1

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Macromolecule #2: TccC3

MacromoleculeName: TccC3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightExperimental: 109 KDa / Theoretical: 109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Insecticidal toxin complex protein TccC3

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Macromolecule #3: TcdB2

MacromoleculeName: TcdB2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightExperimental: 166 KDa / Theoretical: 166 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Insecticidal toxin complex protein TcdB2

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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

BufferpH: 5
Details: 50 mM MES, 100 mM NaCl, 0.05% Tween-20, 5% glycerol
StainingType: NEGATIVE
Details: Grids with adsorbed Protein floated on 0.07% Uranyl Formate for 45 sec.
GridDetails: glow discharged 400 mesh copper grids with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1400
DateSep 29, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 84
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 67210 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 3.4 mm / Nominal defocus max: 0.002 µm / Nominal defocus min: 0.0012 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsPartial-symmetry projection matching approach (after each refinement round, C5 symmetry was applied for the TcdA1 component)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx / Number images used: 3022

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