Three-dimensional strutural study of the sarcoplasmic reticulum clcium ATPase under various physiological conditions.

本研究的目的是揭示在各种生理条件下的钙ATP酶的三维结构，从而阐明主动运输的结构方面。在第一阶段，通过管状晶体的冷冻水化电子显微镜以14 A的分辨率解析无ATP的钙ATP酶的3D结构。在我们发表在《自然》杂志上的论文中，我们描述了跨膜结构是独特的，由三个不同的片段组成;也可以将密度图与从氨基酸序列预测的二级结构相关联。在第二阶段，用ATP的一种不可水解的类似物铬ATP解出了该酶的三维结构，详细比较了两种结构，发现ATP结合口袋的沟被填满了，并且周围的一个结构域移向了沟，好像给结合口袋盖上了盖子。在跨膜段和管腔结构域中也发现了明显的结构变化，表明ATP结合的作用是跨膜传递的。此外，我们还获得了这种酶在高钙浓度下生长的三维微晶的脂双层沿着的视图。虽然图像的数量有限，但分辨率优于10 A。另一方面，开发了一套校正螺旋晶格畸变的程序。它现在已成为可用的，并已被应用于钙ATP酶的管状晶体的图像。经电阻校正后的傅立叶变换效果更好。因此，我们很可能在不久的将来就可以解析这种酶中的单个α-螺旋。

The purpose of this research is to reveal the three-dimensional structure of calcium ATPase under various physiological conditions and thereby elucidate the structural aspects of active transport. At the first stage, 3D structure of calcium ATPase without ATP was solved at 14 A resolution by frozen-hydrated electron microscope of the tubular crystals. In our paper published in Nature, we described that the transmembrane structure is unique, consisting of three distinct segments ; it was also possible to correlate the density map to the secondary structure predicted from the amino acid sequence. At the second stage, three-simensional structure of this enzyme was solved with chromium ATP, a non-hydrolysable analog of ATP.Detailed comparison of two structures revealed that groove predicted to be the ATP binding pocket was filled and one of the surrounding domains has moved towards the groove as if putting a lid to the binding pocket. Distinct structural changes were also found with transmembrance segments and luminal domains, demonstrating that the effect of ATP binding is transmitted across the membrane. Furthermore, we have obtained views along the lipid bilayr of 3D microcrystals of this enzyme grown under a high concentrationof calcium. Though the number of images was limited, the resolution was better than 10 A.On the other hand, a suit of programs has been developed for correcting distortion in helical lattice. It has now become usable and been applied to images of tubular crystals of calcium ATPase. The Fourier tranforms after the sistortion correction appeared much better. Thus, it is very likely that we can resolve individual alpha-helices in this enzyme in a very near future.