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Gating mechanism of the acetylcholine receptor ion channel

乙酰胆碱受体离子通道的门控机制

基本信息

批准号：
06044078
负责人：
TOYOSHIMA Chikashi
金额：
$ 4.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044078/
关键词：
Acetylcholine receptor Ion channel Electron microscopy Structural study Membrane proteins

项目摘要

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.

这项联合研究的最终目标是通过冷冻电子显微镜研究乙酰胆碱受体的三维结构来阐明乙酰胆碱受体的门控机制。具体目标是以更高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。为此，我进行了三次访问，Unwin 博士在支持期间进行了一次访问。第一个访问包括开发根据电子显微照片精确确定 CTF 参数的程序。我们开发了一套在X-window下运行的程序，并将其移植到MRC实验室的计算机上。事实证明，这些程序非常有用且强大。因此，我们在 Unwin 博士的帮助下写了一篇论文，并提交给 Ultramicroscopy。另一个目标是开发用于校正管状晶体晶格中的小变形的程序。我们开发了一种在真实空间中校正它们的方法，Unwin 的小组探索了在倒易空间中针对构成一个重复的 3 个片段细化螺旋轴位置的方法。我们一直在尝试将这两种方法结合起来，以提供更好的校正方案。对于时间分辨实验，已经追求了不同的方法。我们使用笼状化合物的闪光光解，Unwin 使用激动剂（或配体）的喷雾。闪光光解在时间分辨率方面具有优越性，但也不能忽视温度升高的问题。因此，我们在我们的机器中安装了喷涂装置。为了获得足够的喷雾冷冻经验，我准备了许多标本并在英国逗留期间对它们进行了检查。一个实际问题是显微镜网格上发现的管子数量相当少。通过用聚乙二醇粉末浓缩样品解决了这个问题。