STRUCTURE DETERMINATION OF THE NA/K-ATPASE

本项目的目的是研究Na/K-ATPase的结构。 主要的研究工具，电子显微镜和图像处理 它的水晶薄片。在血浆中发现了Na/K-ATPase泵 大多数真核细胞的膜，在那里它在 维持细胞体积，神经和肌肉的兴奋性，以及 在肾脏和肠道吸收。这种酶是 常用于控制心律失常的强心苷。 最初，该酶的三维模型将在 使用三种互补方法获得约2.2 nm的分辨率：a) 从斜视图重建质量分布的三维图像 被负染的水晶片将提供结构 关于从膜上突出的分子结构域的信息。为 Na/K-ATPase，它们占总质量的很大一部分。b) 晶片的冷冻干燥和高分辨率阴影 表面重建方法的使用将提供有关 这两个表面的形态。C)电子显微镜和图像 对片剂的冷冻水合制剂进行处理将会产生结果 与从(A)得到的结果相辅相成，因为它们将揭示结构 酶的跨膜部分的组织。 一项重大努力将致力于对 Na/K-ATPase及其脂质环境。这种酶会被改变 其多肽链和碳水化合物部分的受控消化。 对该酶的消化形式的结构分析将定位其 不同的领域。该酶的脂质环境将被修改为 提高片材的结晶度，从而提高分辨率 超过了目前的2.2纳米。其他物理方法，包括电子 衍射、圆二向色性和低角X射线散射 用来补充电子显微镜的研究。 最终的目标是获得酶的三维结构。 分辨率约为1.0 nm，这将足以解析结构 结构域，如螺旋和对应于通道的配置。 最后，通过将这些结果与有关 序列中，各个结构域的结构可以与它们的作用相关 在运输过程中。

The aim of this project is to study the structure of Na/K-ATPase using, as the principal research tool, electron microscopy and image processing of its crystalline sheets. The Na/K-ATPase pump is found in the plasma membrane of most eukaryotic cells, where it plays a major role in maintenance of cell volume, excitability in nerve and muscle, and absorption in the kidney and intestine. The enzyme is the target of cardiac glycosides that are used routinely for controlling arrhythmias. Initially, a three-dimensional model of the enzyme will be constructed at about 2.2nm resolution using three complementary approaches: a) 3-dimensional reconstruction of the mass distribution from tilted views of the negatively stained crystalline sheets will provide structural information about molecular domains protruding from the membrane. For Na/K-ATPase, these represent a substantial fraction of the total mass. b) Freeze-drying and high resolution shadowing of the crystalline sheets and the use of surface reconstruction methods will supply information about the morphology of the two surfaces. c) Electron microscopy and image processing of frozen hydrated preparations of the sheets will give results complementary to those obtained from (a) as they will reveal the structural organization of the membrane-spanning part of the enzyme. A major effort will be devoted to the biochemical modification of Na/K-ATPase and its lipid environment. The enzyme will be altered by controlled digestion of its polypeptide chains and carbohydrate moiety. Structural analysis of the digested forms of the enzyme will locate its various domains. The lipid environment of the enzyme will be modified to improve the crystallinity of the sheets so as to extend the resolution beyond the current 2.2nm. Other physical methods, including electron diffraction, circular dichroism, and low angle X-ray scattering, will be employed to complement the electron microscopy studies. The ultimate goal is to obtain a 3-dimensional structure of the enzyme at a resolution of about 1.0nm, which would be sufficient to resolve structural domains such as helices and configurations corresponding to channels. Finally, by correlating these results with the information about the sequence, the structure of the various domains can be related to their role in the transport process.