High-resolution single particle electron cryomicroscopy of membrane protein complexes

背景：细胞膜将细胞与其外部环境区分开来。 存在于细胞膜内的蛋白质，称为膜蛋白，作为细胞的门户。 这些蛋白质跨膜运输化学物质，使细胞能够相互交流并感知外部环境。 了解细胞如何进行这些活动需要确定膜蛋白的三维结构。 不幸的是，它往往是困难的或不可能的，以确定膜蛋白的结构，通过传统的方法，如X射线晶体学或核磁共振光谱。 单粒子电子冷冻显微镜（cryo-EM）是一种独特的方法，能够研究大的膜蛋白的结构。 在cryo-EM中，蛋白质被快速冷冻并用电子显微镜成像。 计算图像分析允许将来自许多图像的信息组合成蛋白质的3D图。 我们和其他一两个小组在研究膜蛋白时，已经能够使用单粒子cryo-EM获得~ 1 nm（百万分之一毫米）的分辨率。 在这些分辨率下，解释蛋白质功能的特征开始变得可见。 目的：这项拟议的研究计划是开发新的冷冻EM方法，以提高分辨率，我们可以图像膜蛋白。 该提案的具体目标是：（1）开发获得改进的冷冻EM图像的方法，（2）设计计算更好的三维地图的计算机算法，以及（3）创建解释高分辨率三维地图的新方法。 这些目标将通过研究具有生物重要性的膜蛋白来实现。 重要性：这项研究将为结构生物学的许多研究人员提供工具，并允许在分子水平上深入了解膜蛋白的结构和功能。 通过开发这些工具，我们将打开使用cryo-EM的大门，以更好地了解膜蛋白功能的结构基础。

BACKGROUND: Cell membranes differentiate a cell from its external environment. Proteins that reside within cell membranes, known as membrane proteins, serve as the gateway to cells. These proteins transport chemicals across membranes and allow cells to communicate with each other and sense their external environment. Understanding how cells carry out these activities requires determining the 3-D structures of membrane proteins. Unfortunately, it is often difficult or impossible to determine the structures of membrane proteins by traditional methods such as X-ray crystallography or nuclear magnetic resonance spectroscopy. Single particle electron cryomicroscopy (cryo-EM) is a method uniquely capable of investigating the structures of large membrane proteins. In cryo-EM, proteins are flash frozen and imaged with an electron microscope. Computational image analysis allows information from many images to be combined into a 3-D map of the protein. We, and one or two other groups, have been able to use single particle cryo-EM to obtain resolutions of ~ 1 nm (one millionth of a millimeter) when studying membrane proteins. At these resolutions, the features that explain how the proteins function start to become visible. OBJECTIVE: This proposed research program is for the development of new cryo-EM methods to improve the resolution with which we can image membrane proteins. The specific aims of the proposal are to (1) Develop methods for obtaining improved cryo-EM images, (2) Design computer algorithms for calculating better 3-D maps, and (3) Create new methods for interpreting high-resolution 3-D maps. These aims will be developed by studying membrane proteins of biological importance. SIGNIFICANCE: This research will provide tools for many researchers in structural biology and allow for molecular level insight into the structure and function of membrane proteins. By developing these tools, we will open the door to the use of cryo-EM to gain improved insight into the structural basis of membrane protein function.