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.
