This project involves the determination of the 3-dimensional crystal structure of the channel-forming bacterial toxin Cyt1A and Cry2A produced by the soil bacterium Bacillus thuringiensis (Bt). The proteins from Cyt1A are sequestered as protoxins in crystalline inclusions found within the mass resulting after Bt sporulation. Biorational pesticides consisting of mixtures of Bt endospores and crystalline inclusions are active against dipteran larvae of public health significance (e.g., the encephalitis-bearing Culex mosquito and the malaria-bearing Anopheles mosquito). The mode of action of these toxins involves the formation of ion channels in larval gut epithelial cells which is lethal. The Cyt1A structure will permit the understanding of the body of site-directed mutagenesis data which have identified residues important for toxicity and pore formation and function. The genes coding for these toxins from Cry2A are expressed in susceptible plant parts by insect-resistant transgenic crops. The emergence of resistance to currently deployed Cry toxins and the search for variants with broader susceptible species spectra, optimal potency, and stability properties makes Cry a target for structure-based protein engineering. The Computer Graphics Laboratory is used to visually solve heavy atom Patterson maps leading to initial protein maps. Also, it is used for characterization of map quality, chain tracing, and sequence assignment in electron density maps leading to starting structures subjected to crystallographic refinement. The facilities at the computer graphics laboratory are used for the graphical analysis of these structures.
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