CRYSTAL STRUCTURE OF SAPOSIN B

摘要研究了两种不同蛋白质的晶体。 根据这一提议：皂苷B和BTB结构域。皂苷B(SapB)： 这是在人类溶酶体中发现的一种小蛋白(80个氨基酸)。 通过以下途径激活脑苷硫酸盐以进行代谢分解 芳香族硫酸酯酶A：SapB基因缺陷导致变异 异色性脑白质营养不良，一种致命的脂肪堆积症。 我们研究了来自两种天然来源(猪)的SapB晶体 肾)和重组表达(人SapB在E. Coli.我们已经看到良好的衍射图延伸到 在国际象棋光束线A1的分辨率为2.4埃 猪蛋白。然而，事实证明，这些晶体很难 繁殖，自那以后，我们转向了重组人类蛋白质。 到目前为止，我们只得到了小晶体(0.04 mm x 0.04 mm x 0.02毫米)，反射到10埃 在光束线F2上观察到。我们目前正在优化提纯 和蛋白质的结晶。BTB域：这是一个自治的 在5-10%的锌指转录中发现转录抑制结构域 因子，以及在一些肌动蛋白结合蛋白中。多个BTB域 蛋白质与胚胎发育或癌症有关。我们有 获得具有以下结构域的硒蛋氨酸形式的晶体 在国际象棋F-2中，衍射率为1.9埃。我们收集了一个完整的， 这些晶体上的三个波长的MAD数据集。这些数据是 使用MOSFLM和Scala(Average)的CHESS实现进行处理 Rsym=4.3%，10倍冗余，99%完整性)，以及 用SHELXS确定硒的位置。这一阶段 经过夏普公司的计算，得出的总体数字为 成绩为0.72分。电子密度图异常清晰， 并且我们能够在其中生成域的完整原子模型 计算地图的2天时间。这个结构已经经过了改进 REFMAC，具有出色的统计数据。

Abstract 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.