Crystals from two different proteins have been studied under this proposal: saposin B and the BTB domain. Saposin B (SapB): This is a small (80 amino acid) protein found in the human lysosome that activates cerebroside sulfate for metabolic breakdown by aryslsulfatase A. A genetic deficiency in SapB results in a variant form of metachromatic leukodystrophy, a fatal lipid storage disease. We have studied SapB crystals derived from both natural sources (pig kidney) and from recombinant expression (human SapB expressed in E. coli). We have seen good diffraction patterns extending to a resolution of 2.4 Angstroms at CHESS beamline A1 for crystals of the porcine protein. However, these crystals have proven difficult to reproduce, and we have since turned to the recombinant human protein. To date, we have only obtained small crystals (0.04 mm x 0.04 mm x 0.02 mm) from this material, and reflections to 10 Angstroms have been observed on beamline F2. We are currently optimizing the purification and crystallization of the protein. BTB domain: This is a autonomous transrepression domain found in 5-10% of zinc finger transcription factors, as well as in some actin-binding proteins. Many BTB domain proteins are involved in embryonic development or in cancer. We have obtained crystals of a selenomethionine form of the domain that diffracts to 1.9 Angstrom at CHESS F-2. We have collected a full, three wavelength MAD dataset on these crystals. The data were processed with the CHESS implementation of MOSFLM and SCALA (average Rsym = 4.3% with 10-fold redundancy, 99% completeness), and the selenium positions were determined with SHELXS. The phase calculations were done with SHARP, leading to an overall figure of merit of 0.72. The electron density maps were exceptionally clear, and we were able to generate a full atomic model of the domain within 2 days of calculating the maps. The structure has been refined with REFMAC, with excellent statistics.
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