Computational protocol: Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

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Protocol publication

[…] The data were collected on beamline ID29 at ESRF in Grenoble, France. The diffraction data were processed with MOSFLM and scaled and merged using SCALA . The CCP4 suite of programs was used for all subsequent steps. Molecular replacement was carried out successfully with a number of different hemolysin-like structures. The best results used the S component of S. aureus Panton-Valentine leucocidin (PDB ID 1T5R , as the search model and the program Phaser . This gave a Z-score of 11.6 and a log-likelihood gain of 123.4 following placement of a single copy in the asymmetric unit, and an R-factor and R-free of 41.8 and 44.9% respectively following an initial rigid-body refinement round. [...] Alternating rounds of refinement in Phenix and manual rebuilding in Coot were carried out until no further improvement in R-factor and R-free could be achieved. The final model comprises residues 9 to 290, three Zn ions, three glycerol molecules, one imidazole and 139 water molecules. The final crystallographic R-factor and R-free are 17.9% and 22.8% respectively, and the model has good geometry as assessed by Molprobity . [...] We choose to model a Delta toxin heptamer based on the structure of αHL , for two reasons. Firstly, we excluded a model based on γHL because Delta toxin forms homo-oligomers like αHL, rather than hetero-oligomers as seen in γHL. Secondly, αHL has detectable sequence homology to Delta toxin, whereas the more recently solved (and structurally related) Vibrio cholerae cytolysin heptamer has inserted domains and much lower sequence homology. Delta toxin was sequence aligned with a number of other known hemolysin-like atomic structures using ClustalW and αHL (PDB ID 7AHL) , and residues corresponding to the stem and latch domains in αHL were deleted from the final refined model of Delta toxin. The coordinates of the refined Delta toxin minus these two regions were then optimally superposed onto the A molecule from αHL using SSM in CCP4. The residues corresponding to the latch and stem domains from 7AHL where mutated to their corresponding residues in Delta toxin using Chainsaw from CCP4 and these two domains added to the Delta toxin. This model of a Delta toxin pore-form monomer was then superposed on each of the 7 monomers in 7AHL in turn to form a model of a heptameric Delta toxin pore. The model was then energy-minimised in Phenix . […]

Pipeline specifications

Software tools iMosflm, CCP4, PHENIX, Coot, MolProbity, Clustal W
Applications Small-angle scattering, Protein structure analysis
Organisms Clostridium perfringens, Homo sapiens, Staphylococcus aureus
Diseases Enteritis, Drug-Related Side Effects and Adverse Reactions
Chemicals Glycerol