Structural Basis of Active Transport by Ion Pumps

本项目的目的是通过分析离子泵在输运周期中不同阶段的三维结构来阐明活性离子输运的结构基础。我们一直在研究肌浆网钙atp酶，它是p型离子泵的代表成员，负责从细胞质中泵出钙离子来放松肌肉细胞。由于只能形成管状和非常薄的三维晶体，我们已经付出了很多努力来改进图像分析技术。日本方面开发了螺旋分析程序，并将其与安装在纽约大学的新型显微镜相结合，研究了具有CrATP或thapsignargin束缚的管状晶体的结构。结果，我们成功地确定了atp结合位点（Biopys.J.）。，已出版)，并取得8 *分辨率的地图。在这些图中，可以看到10个跨膜螺旋和离子通路（Nature, In press）。只有通过国际合作才能取得这些良好成果，并进行了4次国际旅行。至于在钙的存在下形成的三维晶体，还需要大量的努力来建立方法学。目前，已经获得了一张9 *分辨率的图，显示了沿脂质双层的晶体视图。这种结构与在没有钙的情况下形成的管状晶体的结构大不相同。这项工作的手稿已提交给Biohys.J。

The goal of this project is to elucidate the structural basis of active ion transport by analysing the three-dimensional structures of ion pumps at various stages in the transport cycle. We have been working on calcium ATPase of sarcoplasmic reticulum, which is a representative member of P-type ion pumps and responsible for relaxing muscle cells by pumping calcium ions from cytoplasm. Since only tubular and very thin three-dimensional crystals can be formed, we have put much efforts to improve the technology for image analysis. Japan side has developed programs for helical analysis and combined them with a new microscope installed in New York University to study the structures of the tubular crystals with CrATP or thapsigargin bound. As a result, we have succeeded in determining the ATP-binding site (Biopys.J., publised) and obtaining 8 * resolution maps. In these maps, 10 transmembrane helices and ion pathway can be seen (Nature, in press). These good results have been possible only with international collaboration, and 4 international travels have been made. As to the three-dimensional crystals formed in the presence of calcium, much efforts are still needed for establishing the methodology. At present, a 9 *-resolution map has been obtained showing the view of the crystal along the lipid bilayr. The structure is much different from that obtained from the tubular crystals formed in the absence of calcium. The manuscript on this work has been submitted to Biohys.J.