Gating mechanism of the acetylcholine receptor ion channel

本联合研究的最终目的是通过低温电子显微镜研究乙酰胆碱受体的三维结构来阐明其门控机制。具体目的是以更高的分辨率分析管状晶体，并可视化不同生理状态下受体的三维结构。为了做到这一点，我去了三次，在我支持的时间段内，Unwin博士去了一次。第一部分包括从电子显微照片中精确测定CTF参数的程序的开发。我们开发了一套在x窗口下运行的程序，并将它们移植到MRC实验室的计算机上。这些程序已被证明是非常有用和健壮的。因此，我们在Unwin博士的帮助下写了一篇论文，并提交给了Ultramicroscopy。另一个目标是开发用于纠正管状晶体晶格中的微小畸变的程序。我们已经开发了一种在真实空间中纠正它们的方法，Unwin的团队已经探索了在互反空间中为构成一个重复的3段精炼螺旋轴位置的方法。我们一直在尝试将两种方法结合起来，以提供更好的校正方案。对于时间分辨实验，人们采用了不同的方法。我们使用闪光光解笼状化合物和Unwin使用喷雾激动剂（或配体）。闪光光解在时间分辨率方面具有优势，但不能忽视温升问题。因此，我们在我们的机器上实现了喷涂装置。为了获得足够的冷冻经验，我在英国期间准备了许多标本并进行了检查。一个实际问题是，在显微镜网格上发现的管的数量相当少。用聚乙二醇粉末浓缩试样，解决了这一问题。

The ultimate goal of this joint research is to elucidate the gating mechanism of the acetylcholine receptor by studying the three-dimesional structure by cryo-electron microscopy. The specific aims were to analyze the tubular crystals at a higher resolution and to visualise the three-dimensional structure of the receptor in different physiological states. To do so, I made three visits and Dr Unwin made one for the the period supported.The first one includes the development of the programs for determining the CTF parameters precisely from electron micrographs. We have developed a set of programs running under the X-window and ported them to the computers at the MRC laboratory. These programs have proven to be very useful and robust. Therefore, we have written a paper, with the help of Dr Unwin, and submitted to Ultramicroscopy.The other aim was to develop programs for correcting small distortions in crystal lattice of a tubular crystal. We have developed a method to correct them in real space and Unwin's group have explored the way in which the helix axis position was refined in reciprocal space for 3 segments constituting one repeat. We have been trying to combine both ways to provide better correction scheme.For time-resolving experiments, different methods have been pursued. We use flash photolysis of caged-compounds and Unwin uses spraying of agonist (or ligand). Flash photolysis is superior in terms of timeresolution but it cannot neglect the problem of temperature rise. Hence we are implementing the spraying apparatus in our machine. To acquire enough experience with spary freezing I prepared many specimens and examined them during my stay in England. One practical problem was that the numbers of tubes found on microscope grids were rather small. This problem was solved by concentrating the specimen by powder of polyethylen glycol.