EMDB-2297: Structure of TcdA1 in prepore state

Basic information

• Entry: Database: EMDB / ID: EMD-2297
• Title: Structure of TcdA1 in prepore state
• Map data: Reconstruction of the TcdA1 prepore complex

Sample

• Sample: TcdA1
• Protein or peptide: TcdA1

• Keywords: Photorabdus / translocation / pore formation / bacterial toxin / TcA / helical toxin

Function / homology

Function:

identical protein binding

Domain/homology:

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

Component:

TcdA1

• Biological species: Photorhabdus luminescens (bacteria)
• Method: single particle reconstruction / cryo EM / Resolution: 6.3 Å
• Authors: Gatsogiannis C / Lang A E / Meusch D / Pfaumann V / Hofnagel O / Benz R / Aktories K / Raunser S
• Citation: Journal: 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 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

Deposition	Jan 25, 2013	-
Header (metadata) release	Feb 13, 2013	-
Map release	Feb 13, 2013	-
Update	Apr 3, 2013	-
Current status	Apr 3, 2013	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_2297.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstruction of the TcdA1 prepore complex
• Voxel size: X=Y=Z: 1.25 Å

Density

Contour Level	By AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum	-1.07592964 - 2.55297446
Average (Standard dev.)	0.01785584 (±0.12799375)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -150 -150 -150 Dimensions 300 300 300 Spacing 300 300 300
Cell	A=B=C: 375.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.25	1.25	1.25
M x/y/z	300	300	300
origin x/y/z	0.000	0.000	0.000
length x/y/z	375.000	375.000	375.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-36	-30	-80
NX/NY/NZ	73	61	161
MAP C/R/S	1	2	3
start NC/NR/NS	-150	-150	-150
NC/NR/NS	300	300	300
D min/max/mean	-1.076	2.553	0.018

Supplemental data

Sample components

Entire : TcdA1

• Entire: Name: TcdA1

Components

• Sample: TcdA1
• Protein or peptide: TcdA1

Supramolecule #1000: TcdA1

• Supramolecule: Name: TcdA1 / type: sample / ID: 1000 / Oligomeric state: Pentamer / Number unique components: 1
• Molecular weight: Experimental: 1.41 MDa / Theoretical: 1.41 MDa

Macromolecule #1: TcdA1

• Macromolecule: Name: 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 weight: Experimental: 283 KDa / Theoretical: 283 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: CodonPlus; Recombinant plasmid: pCR-BluntII-TOPO, subcloned into pET-28a(+)
• Sequence: UniProtKB: TcdA1

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.02 mg/mL
• Buffer: pH: 5 ; Details: 100 mM NaCl, 0.05% Tween-20, 50 mM MES , 5% glycerol
• Grid: Details: C-Flat 1.2/1.3-4C copper 400 mesh, with additional thin carbon support
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 1 sec before plunging

Electron microscopy

• Microscope: JEOL 3200FSC
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 124472 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 0.0024 µm / Nominal defocus min: 0.0005 µm / Nominal magnification: 60000
• Specialist optics: Energy filter - Name: in-column Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 15.0 eV
• Sample stage: Specimen holder model: JEOL 3200FSC CRYOHOLDER
• Alignment procedure: Legacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
• Date: Nov 2, 2011
• Image recording: Category: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 251 / Average electron dose: 20 e/Ų

Image processing

• CTF correction: Details: each particle
• Final reconstruction: Applied symmetry - Point group: C₅ (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx / Number images used: 45158