Electron Crystallography of Ca++ - ATPase from Sarcoplasmic Reticulm

肌浆网钙-ATPase转运钙离子是肌肉生理学中的一个基本过程。这种酶将胞浆中的钙离子移至肌浆网中的储存位置，从而导致肌肉松弛。本项目致力于对洗涤剂溶解的酶在高钙浓度下产生的大的多层晶体阵列进行电子结晶学分析。提出的研究将1.发展从不同厚度的多层晶体中获得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保持三维有序的方案，3.研究有利于产生无堆叠的2-D薄片的结晶方案，以便于在更高分辨率下进行电子结晶学分析，4.通过寻找颗粒晶体X射线图中α螺旋、β片层和脂类的特征衍射图，获得有关钙-ATPase分子的蛋白质二级结构的信息。将努力进行这种结构分析，至少达到蛋白质二级结构(约7A)的分辨率，以测试现有的钙-ATPase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.