Structure Analysis of the Acetylcholine Receptor in Tubular Membrane Crystals

描述(申请人提供)：许多膜蛋白、可溶性蛋白和人造蛋白质-脂复合体在近生理条件下倾向于组装成管状晶体。这类标本是电子显微镜结构和功能研究的理想标本，我们最近的研究证明了这一点，该研究导致了烟碱型乙酰胆碱(ACh)受体的精炼原子模型。拟议研究的目的是：(A)通过确定开放通道形式的受体的高分辨率结构来补充对闭合通道形式的受体的分析，从而了解原子尺度下的门控的结构基础；(B)改进从管状晶体中求解结构的方法，以便能够快速和最少地从各种管状晶体中推导出蛋白质组件的近原子模型。在发生显著的脱敏反应之前，将使用快速喷雾冷冻技术来实现适当的短反应时间(~5ms)，并将通道捕获在打开状态。成像将在液氦温度下进行，以将辐射损害降至最低，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，这些例程将与在Scripps工作的相关标本的同事合作实现自动化；这将使快速筛选最佳图像成为目前限制高分辨率结构确定速度的主要步骤。了解ACh受体的门控机制(以及其他递质门控离子通道)与许多神经疾病有关，因为这是神经系统所有快速突触通讯的基本过程。我们使用和正在开发的冷冻电子结晶学方法是重要的，因为它是在天然脂环境和近生理离子条件下分析膜蛋白结构的最有效的方法。

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.