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- EMDB-2298: Structure of TcdA1 pore (TcdA1 reconstituted in liposomes) -

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Basic information

Entry
Database: EMDB / ID: EMD-2298
TitleStructure of TcdA1 pore (TcdA1 reconstituted in liposomes)
Map dataReconstruction of TcdA1 reconstituted in liposomes
Sample
  • Sample: TcdA1
  • Protein or peptide: TcdA1
KeywordsPhotorabdus / translocation / pore formation / bacterial toxin / TcA / helical toxin
Function / homology
Function and homology information


identical protein binding
Similarity search - Function
: / 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 ...: / 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
Similarity search - Domain/homology
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.9 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: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2298.png

Downloads & links

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Map

FileDownload / File: emd_2298.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TcdA1 reconstituted in liposomes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.65 Å/pix.
x 128 pix.
= 467.2 Å		3.65 Å/pix.
x 128 pix.
= 467.2 Å		3.65 Å/pix.
x 128 pix.
= 467.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.03510948 - 0.07465743
Average (Standard dev.)0.00113197 (±0.00600149)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 467.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.653.653.65
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z467.200467.200467.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0350.0750.001

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Supplemental data

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Sample components

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Entire : TcdA1

EntireName: TcdA1
Components
  • Sample: TcdA1
  • Protein or peptide: TcdA1

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Supramolecule #1000: TcdA1

SupramoleculeName: TcdA1 / type: sample / ID: 1000 / Oligomeric state: Pentamer / Number unique components: 1
Molecular weightExperimental: 1.41 MDa / Theoretical: 1.41 MDa

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Macromolecule #1: TcdA1

MacromoleculeName: TcdA1 / type: protein_or_peptide / ID: 1 / Name.synonym: Toxin A / Details: Protein reconstituted in liposomes / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 11 / Details: 50 mM CAPS pH 11, 100 mM NaCl, 5 % glycerol
GridDetails: C-Flat 1.2/1.3-4C copper 400 mesh, with additional thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 1 sec before plunging

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Electron microscopy

MicroscopeJEOL 3200FSC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
Specialist opticsEnergy filter - Name: in-column Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 15.0 eV
DateMar 18, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 145 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 85460 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm / Nominal defocus max: 0.005 µm / Nominal defocus min: 0.0029 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER

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Image processing

CTF correctionDetails: Each Particle
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx / Number images used: 5549

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