Structural elucidation of active ion transport

这个项目的目标是从结构标准点汲取主动运输的机制。当这个项目被提出时，我们有两种幸运的晶体Ca-D12 + I-D1-ATP酶用于电子显微镜。自那时以来，我们成功地开发了一个适用于X射线晶体学的大型晶体，并有望在两年内解决结构的原子分辨率。因此，我们对利用结构研究和要求教授的突变体的压力产生了怀疑。马里兰大学的Inesi加入了，因为他是唯一一个这样的群体，成功地产生了这种膜蛋白质的大数量。这个合作非常好，并制作了一份评论和一份原始研究论文（提交给生物化学）。与X射线晶体学（提交给自然）揭示的结构的相互研究一致得很好，并提供了对主动传输所要求的规范性变化的见解。在另一方面，该结构是可以独立地解释正确的生物化学和相互作用结果的。Altogether，这个项目是一个很好的国际合作范例。下一个步骤显然是制造突变体的水晶。由于这一目的，SR Cai-D12 + Yi-D1的净化程序已经被转化为不充分的。We are now improving the methods for cultured cells in collaboration with Prof. Inesi.

The aim of this project is to elucidate the mechanism of active transport from the structural standpoint. When this project was proposed, we had merely two kinds of crystals of CaィイD12+ィエD1-ATPase useful for electron microscopy. Since then, we succeeded in generating large crystals suitable for X-ray crystallography and expected to solve the structure at an atomic resolution within two years or so. Therefore, we shifted the stress on considering on the utilization of mutants for structural studies and asked Prof. Inesi of the University of Maryland to join, because his group was the only one that-succeeded in making large quantity of this membrane protein. This collaboration went out extremely well and produced one review and one original research paper (submitted to Biochemistry). The mutational studies agreed very well with the structure revealed by x-ray crystallography (submitted to Nature) and provided insights into the conformational changes required for active transport. On the other hand, the structure was indispensable to interpret the biochemical and mutational results correctly. Altogether, this project became a very good example of international collaboration. The next step is obviously to make crystals of mutants. For this purpose, the purification procedure used for SR CaィイD12+ィエD1-ATPase has turned out to be insufficient. We are now improving the methods for cultured cells in collaboration with Prof. Inesi.