Krebs Angelika, Edwards Patricia C, Villa Claudio, Li Jade, Schertler Gebhard F X
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
J Biol Chem. 2003 Dec 12;278(50):50217-25. doi: 10.1074/jbc.M307995200. Epub 2003 Sep 25.
G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22(1)2(1) symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45 degrees. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 A in the membrane plane and approximately 13 A perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the beta-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified.
G蛋白偶联受体是整合膜蛋白，可响应环境信号并启动信号转导途径，从而激活细胞过程。视紫红质是G蛋白偶联受体家族的一个著名成员，位于视杆外段的盘状膜中，负责暗光视觉。视紫红质是研究最广泛的G蛋白偶联受体，关于其结构的知识为其他相关受体提供了模板。我们通过2000年使用三维晶体的X射线晶体学解析得到的晶体结构（1）获得了详细的结构知识。在此，我们报告了通过对具有p22(1)2(1)对称性的二维晶体进行电子冷冻显微镜测定得到的牛视紫红质的三维密度图。使用相对较小且无序的晶体使得结构测定过程具有挑战性。由于可用的原始数据限于最大45度的倾斜度，因此特别关注了振幅和相位的提取。在精修过程中，应用了改进的拉直程序。这导致在膜平面上的最终分辨率为5.5埃，垂直于膜平面约为13埃，使我们的电子密度图成为目前通过电子显微镜获得的最精确的G蛋白偶联受体图。最重要的是我们获得的关于膜平面中心以及分子相对于双层的取向的信息。这些信息无法从三维晶体中获取。在我们的电子密度图中，识别出了所有七个跨膜螺旋，它们的排列与从晶体结构（1）中已知的排列一致。在视网膜结合口袋中，与螺旋3相邻的一个密度峰表明了发色团的β-紫罗兰酮环的位置，并且在其附近可以识别出几个较大的氨基酸。
