Electron Crystallography of Ca++ - ATPase from Sarcoplasmic Reticulm

肌浆网 Ca2-ATP 酶进行的 Ca2 离子转运是肌肉生理学的基本过程。 这种酶将 Ca2 从细胞质转移到肌浆网的储存位点，从而导致肌肉松弛。 该项目的重点是在高 Ca2 浓度存在下，对由去污剂溶解的酶产生的大型多层晶体阵列进行电子晶体学分析。 拟议的研究将 1. 开发从不同厚度的多层晶体获取和组合电子衍射图像数据的方法，2. 优化用于电子显微镜分析的晶体中 3-D 顺序保存的方案，3. 研究有利于产生无堆叠的 2-D 片层以促进更高分辨率的电子晶体分析的结晶方案，以及 4. 通过搜索 α 螺旋、β 片层和颗粒晶体的 X 射线图中的脂质。 我们将努力将该结构分析至少解析到蛋白质二级结构（大约7A），以测试当前的Ca2-ATP酶结构模型。

Ca2+ ion transport by the Ca2+-ATPase of sarcoplasmic reticulum is a fundamental process in muscle physiology. This enzyme removes Ca2+ from the cytoplasm to storage sites in the sarcoplasmic reticulum resulting in muscle relaxation. This project focusses on electron crystallographic analysis of large multilayered crystalline arrays produced from detergent solubilized enzyme in the presence of high Ca2+ concentrations. The studies proposed will 1. develop methodology for obtaining and combining electron diffraction image data from multilayered crystals of different thickness, 2. optimize the protocols for preservation of 3-D order in the crystals for electron microscopic analysis, 3. investigate crystallization protocols that favor the production of 2-D sheets free from stacking to facilitate the electron crystallographic analysis at higher resolution, and 4. obtain information on protein secondary structure of the Ca2+-ATPase molecule by searching for characteristic diffraction from alpha helices, beta sheets and lipid in X-ray diagrams of pelleted crystals. Efforts will be made to carry this structure analysis at least to the resolution of protein secondary structure (approximately 7A) to test current models of Ca2+-ATPase structure.