High-resolution single particle electron cryomicroscopy of membrane protein complexes

背景：细胞膜是细胞与其外部环境的区别。细胞膜内的蛋白质，被称为膜蛋白，是进入细胞的门户。这些蛋白质通过细胞膜运输化学物质，使细胞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，通常很难或不可能通过传统的方法，如x射线晶体学或核磁共振波谱学来确定膜蛋白的结构。单粒子电子低温显微镜（cryo-EM）是一种独特的研究大型膜蛋白结构的方法。在低温电子显微镜下，蛋白质被快速冷冻，并用电子显微镜成像。计算图像分析可以将来自许多图像的信息组合成蛋白质的三维地图。在研究膜蛋白时，我们和其他一两个小组已经能够使用单粒子冷冻电镜获得~ 1纳米（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清晰可见。

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.