Crystallographic study of calcium ion pump of sarcoplasmic reticulum

为阐明肌浆网主动转运的结构基础，对肌浆网钙离子泵(Ca^&lt；2+&Gt；-ATPase)进行了结晶学研究。当我们开始这个项目时，我们已经知道了两种类型的CA^&lt；2+&Gt；-ATPase晶体：一种是在EGTA和钒酸盐存在下形成的天然SR膜的管状晶体；另一种是在高浓度钙存在下形成的由重组膜片组成的小的三维晶体。这两种材料都很小，适合电子结晶学。使用管状晶体，在8A分辨率下获得了三种状态的三维结构：无ATP，有铬-ATP和有thapsigarin(最后一个是与纽约大学Stokes实验室合作)。与thapsigargin结合的那个得到了最好的解析，并清楚地显示了从二级结构预测中预期的10个跨膜螺旋(发表在《自然》杂志上)。含铬的ATP指示了ATP结合位点的位置(发表在《生物物理学》上)。J.)。我们为此开发了一套广泛的计划。比较小的3D晶体和管状晶体的3D结构沿b轴的投影，发现由于钙(生物物理)的结合，发生了大规模的构象变化。通过改进晶化条件，我们成功地制备出了有序的片状离子泵晶体。最薄的可以用电子分析，最厚的可以用X射线结晶学分析。我们已经开发了一台用于收集电子衍射强度的旋转相机，并了解到即使是最薄的晶体也与最厚的晶体具有几乎相同的晶格常数。因此，我们开始直接从电子显微照片中收集相信息，并获得了某些反射的一组非常有用的相。我们现在正在寻找良好的同构重金属衍生物，以便在原子分辨率下解决结构问题。

Crystallographic study of the calcium ion pump (Ca^<2+>-ATPase) of sarcoplasmic reticulum(SR)has been performed to elucidate the structural basis of active transport. Two types of crystal of CA^<2+>-ATPase were known when we started this project : one was tubular crystal of native SR membrane formed in the presence of EGTA and vanadate ; the other was small three-dimensional crystals consisting of reconstituted membrane sheets formed in the presence of high concentration of calcium. Both were small and suitable for electron crystallography. Using tubular crystals, three-dimensional structures were obtained for three states at 8 A resolution : no ATP, with chromium-ATP and with thapsigargin (the last one was in collaboration with Stokes Lab at NY University). The one with bound thapsigargin was best resolved and clearly showed 10 transmembrane helices expected from secondary structure predictions (published in Nature). The one with chromium ATP indicated the position of ATP-binding site(published in Biophys. J.). We have developed an extensive set of programs for this purpose. Comparison of the projection along the b-axis of the small 3D crystals and 3D structure from tubular crystals revealed large scale conformational changes attributable to the binding of calcium (Biophys. J.).By improving the crystallisation conditions, we succeeded in preparing well ordered plate-like crystals of this ion pump. The thinnest ones could be analysed by electron and the thickest ones by X-ray crystallography. We have developed a rotation camera for collecting electron diffraction intensities and learned that even the thinnest crystals have virtually the same lattice constants with the thickest ones. We therefore proceeded to collect phase information directly from the electron micrographs and obtained a very useful set of phases for certain reflections. We are now looking for good isomorphous heavy metal derivatives for solving the structure at an atomic resolution.