FROZEN HYDRATED ELECTRON MICROSCOPY OF CA ATPASE

提供依赖于ATP的各种离子在 多种细胞膜和亚细胞膜。这些泵负责 对于细胞静息电位(Na+/K+-ATPase)等重要现象 和肌肉松弛(钙-ATPase)。钙泵(钙-ATPase)有 是这个家庭的原型，每个人都有 可以想到的方法，包括动力学、光谱学、现场定向 诱变和化学修饰。我们对分子的理解 然而，由于我们对分子结构的忽视，这一机制受到了阻碍。 这项提议旨在用电子方法确定这种结构。 冰冻水合钙-三磷酸腺苷酶晶体的结晶学研究 骨骼肌肌浆网。具体地说，两种晶型 正在研究中。提纯的、薄的、多层的晶体， 洗涤剂增溶的Ca~(2+)-ATPase衍射率高 通过修改，提出了6A分辨率下的三维结构 二维电子结晶学标准方法的发展 膜蛋白。肌浆网膜中的管状晶体 以前被用于14A结构和组织的 将通过标记钙-ATPase来进一步研究分子 并在3D重建中定位这些标记。 管状晶体的结构分辨率也将得到提高 通过使用改进的电子显微镜设备和改进的策略 用于图像分析。这两种晶型的结构代表 Ca~(2+)-ATPase的不同构象状态，对应于主要 反应周期中的中间体。因此，比较所产生的 结构将有助于理解偶联ATP的结构基础 水解到钙的运输。给定氨基酸中的同源关系 顺序和反应机理的相似性，这些结论将 更广泛地应用于P型离子泵家族的其他成员(例如， Na+/K+-ATPase，H+/K+-ATPase)，并帮助开发一种通用的机制 依赖于ATP的离子转运。在铜运输的情况下，缺陷 导致门克斯或威尔逊病，更好地理解 这一机制最终可能有助于制定治疗策略。

DESCRIPTION providing ATP-dependent transport of various ions across a variety of cellular and subcellular membranes. These pumps are responsible for such important phenomena as the cell resting potential (Na+/K+-ATPase) and muscle relaxation (Ca2+-ATPase). The calcium pump (Ca2+-ATPase) has been an archetype for this family and has been characterized by every conceivable means, including kinetics, spectroscopy, site-directed mutagenesis and chemical modification. Our understanding of the molecular mechanism, however, is hindered by our ignorance of the molecular structure. This proposal aims to determine this structure by methods of electron crystallography employing frozen-hydrated crystals of Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. In particular, two crystal forms are being studied. Thin, multilamellar crystals of purified, detergent-solubilized Ca2+-ATPase diffract to high resolution and a three-dimensional structure at 6 A resolution is proposed by modifying standard electron crystallographic methods developed for two-dimensional membrane proteins. Tubular crystals in the sarcoplasmic reticulum membrane have previously been used for a 14 A structure and the organization of the molecule will be further investigated by labelling Ca2+-ATPase with site-specific compounds and locating these labels in 3D reconstructions. The resolution of the structure from tubular crystals will also be improved by using improved facilities for electron microscopy and improved strategies for image analysis. Structures from these two crystal forms represent different conformational states of Ca2+-ATPase, corresponding to major intermediates in the reaction cycle. Thus, comparison of the resulting structures will help to understand the structural basis for coupling ATP hydrolysis to calcium transport. Given the homologies in amino acid sequence and similarities in reaction mechanisms, these conclusions will apply more broadly to other members of the family of P-type ion pumps (e.g., Na+/K+-ATPase, H+/K+-ATPase) and help develop a general mechanism for ATP-dependent ion transport. In the case of copper transport, deficiencies which lead either to Menkes or Wilson disease, a better understanding of this mechanism may eventually help in developing strategies for treatment.