CRYSTAL STRUCTURE OF SAPOSIN B

SAPOSIN B 的晶体结构

• 批准号: 6120523
• 负责人: GIL PRIVE
• 金额: $ 0.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120523
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

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.

摘要已经研究了两种不同蛋白质的晶体 根据该提案：saposin 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） 激活硫酸脑苷脂进行代谢分解 芳基硫酸酯酶 A。SapB 的遗传缺陷导致变异 异染性脑白质营养不良的一种形式，一种致命的脂质储存疾病。 我们研究了两种天然来源（猪 肾）和重组表达（在大肠杆菌中表达的人 SapB） 大肠杆菌）。 我们已经看到良好的衍射图案延伸到 CHESS 光束线 A1 的晶体分辨率为 2.4 埃 猪蛋白。 然而，这些晶体已被证明很难 繁殖，从那时起我们就转向重组人类蛋白质。 迄今为止，我们仅获得了小晶体（0.04 mm x 0.04 mm x 0.02 毫米）从这种材料，和反射到 10 埃已经 在光束线 F2 上观察到。 我们目前正在优化纯化 和蛋白质的结晶。 BTB 域：这是一个自治域 5-10% 的锌指转录中发现反式阻抑结构域 因子以及一些肌动蛋白结合蛋白。 许多BTB域名 蛋白质参与胚胎发育或癌症。 我们有 获得了硒代蛋氨酸结构域的晶体 在 CHESS F-2 中衍射为 1.9 埃。 我们收集了完整的、 这些晶体的三波长 MAD 数据集。 数据是 使用 MOSFLM 和 SCALA 的 CHESS 实现进行处理（平均 Rsym = 4.3%，具有 10 倍冗余，完整性为 99%），并且 硒位置由 SHELXS 确定。 阶段 使用 SHARP 进行计算，得出的总体数字为 优点为0.72。 电子密度图异常清晰， 我们能够生成该域的完整原子模型 2天计算地图。 该结构已被细化 REFMAC，具有出色的统计数据。