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-脂质复合物在接近生理条件下优先组装成管状晶体。这样的标本是理想的结构和功能的电子显微镜研究，我们最近的研究表明，导致一个精致的原子模型的烟碱乙酰胆碱（ACh）受体。拟议研究的目的是：（a）通过确定开放通道形式受体的高分辨率结构来补充对封闭通道形式受体的分析，从而了解门控的结构基础原子尺度细节;（B）改进用于从管状晶体求解结构的方法，使得近-蛋白质组装体的原子模型可以从各种管状晶体中快速且以最小的努力导出。将使用快速喷雾冷冻技术来实现适当的短暂（约5 ms）反应时间，并在发生显著脱敏之前将通道捕获在开放状态。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例程，这些例程将与Scripps的相关标本工作的同事合作自动化;这将使快速筛选最佳图像成为可能，这是目前限制高分辨率结构确定速率的主要步骤。对乙酰胆碱受体（以及其他递质门控离子通道）门控机制的理解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讯的基础过程。我们使用和正在开发的冷冻电子晶体学方法是重要的，因为它是最强大的方法，可用于分析膜蛋白在其天然脂质环境和近生理离子条件下的结构。

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.