Structure Analysis of the Acetylcholine Receptor in Tubular Membrane Crystals

描述（由申请人提供）：许多膜蛋白，可溶性蛋白和人工蛋白-脂复合物在接近生理条件下倾向于组装成管状晶体。这些标本是通过电子显微镜进行结构和功能研究的理想选择，正如我们最近的研究所证明的那样，导致了尼古丁乙酰胆碱（ACh）受体的精细原子模型。本研究的目的是：(a)通过确定开通道形式受体的高分辨率结构来补充对闭通道形式受体的分析，从而获得对门控在原子尺度细节上的结构基础的理解；(b)改进从管状晶体中求解结构的方法，使蛋白质组装的近原子模型可以从各种管状晶体中快速、省力地得到。快速喷雾冷冻技术将用于实现适当的短暂（~5毫秒）反应时间，并在显着脱敏发生之前将通道困在打开状态。成像将在液氦温度下进行，以尽量减少辐射损伤并优化电子光学性能。图像处理将利用以前开发的和新的程序，这些程序将与在斯克里普斯研究相关标本的同事合作实现自动化；这将使最佳图像的快速筛选成为可能，这是目前限制高分辨率结构确定率的主要步骤。了解ACh受体的门控机制（以及其他递质门控离子通道）与许多神经系统疾病有关，因为它是神经系统中所有快速突触通信的基本过程。我们正在使用和开发的低温电子晶体学方法非常重要，因为它是分析膜蛋白在天然脂质环境和近生理离子条件下结构的最有效方法。

DESCRIPTION (provided by applicant): Many membrane proteins, soluble proteins and artificial protein-lipid complexes have a preference to assemble into tubular crystals under near-physiological conditions. Such specimens are ideal for structural and functional studies by electron microscopy, as was demonstrated by our recent study leading to a refined atomic model of the nicotinic acetylcholine (ACh) receptor. The purpose of the proposed research is: (a) to complement that analysis of the receptor in the closed-channel form by determining the high resolution structure of the receptor in the open-channel form, and hence obtain an understanding of the structural basis of gating in atomic-scale detail; (b) to improve the methodology for solving structures from tubular crystals so that near-atomic models of protein assemblies can be derived from all kinds of tubular crystal quickly and with minimum effort. A rapid spray-freezing technique will be used to achieve an appropriate brief (~5 ms) reaction time and trap the channels in the open state, before significant desensitization can take place. The imaging will be conducted at liquid helium temperatures to minimize radiation damage and to optimize electron-optical performance. The image processing will make use of previously developed and new routines, which will be automated in collaboration with colleagues working on related specimens at Scripps; this will enable rapid screening for the best images, which is the main step at present limiting the rate of high resolution structure determination. An understanding of the gating mechanism of the ACh receptor (and hence of other transmitter-gated ion channels) is relevant to many kinds of neurological disorder, because it is the fundamental process underlying all fast synaptic communication in the nervous system. The cryo-electron crystallographic approach we use and are developing, is important because it is the most powerful method available to analyze the structures of membrane proteins in their native lipid environment and under near-physiological ionic conditions.