Atomic Resolution in Biological Electron Microscopy

描述（由申请人提供）：该项目的广泛目标，现在进入第十个年头，是在生物应用范围不断扩大的背景下开发电子低温显微镜（cryoEM）的实验和计算工具。我们特别寻求将电子显微镜（EM）与X射线晶体学联系起来，并将分子EM推向成为一种高分辨率的工具。在过去的资助期间，几项进展之一是对几种病毒结构达到了近原子分辨率（<4 A），实现了亨德森15年前提出的猜想，即有可能通过冷冻电镜在这种细节水平上对生物组装体进行成像。我们为下一个项目期间提出了三个主要主题。(1)方法的不断发展。我们将扩展近原子分辨率结构的计算方法，以包括多态单粒子和螺旋组件的图像（Grigorieff，Harrison）;我们将改进样品制备的均匀性和均匀性，建立在最后一个资助期（Walz）的亲和网格的发展基础上;我们将探索减少光束诱导运动的方法（Grigorieff）;细胞成像的分辨率改进（Nicastro）。(2)电子冷冻断层扫描（cryo-ET）作为近原子分辨率结构可视化与通过光学显微镜和活细胞成像研究其细胞内动力学之间的桥梁（Nicastro，Harrison，Grigorieff）。轮状病毒进入和网格蛋白外壳动力学是两个具体的项目，其结构被确定为该项目的一部分，并从现场获得结果。 细胞荧光显微镜提出的机制问题，最好回答的前沿方法在冷冻ET。(3)瞬态和多状态程序集的分析，包括作为主题1的一部分开发的方法所带来的增强功能（所有四个项目）。在追求这一主题，我们将特别关注动态结构的大规模组织，如动粒，纤毛和运输囊泡拴系复合物。

DESCRIPTION (provided by applicant): The broad goals of this Project, now entering its tenth year, are to develop the experimental and computational tools of electron cryomicroscopy (cryoEM) in the context of a widening range of biological applications. We seek in particular to connect electron microscopy (EM) with x-ray crystallography and to move molecular EM toward becoming a high-resolution tool. One of several advances during the past funding period has been to reach near-atomic resolution (<4A) for several virus structures, fulfilling a conjecture made 15 years ago by Henderson that it would be possible to image biological assemblies by cryoEM at this level of detail. We propose three principal themes for the coming project period. (1) Continued methods development. We will extend computational methods for near-atomic resolution structures to include images of multi-state single particles and helical assemblies (Grigorieff, Harrison); we will improve sample preparation for uniformity and homogeneity, building on the development of Affinity Grids during the last grant period (Walz); we will explore methods to reduce beam-induced movement (Grigorieff); resolution improvement for cellular imaging (Nicastro). (2) Electron cryotomography (cryo-ET) as a bridge between visualizing near-atomic resolution structures and studying their intracellular dynamics by optical microscopy and live-cell imaging (Nicastro, Harrison, Grigorieff). Rotavirus entry and clathrin-coat dynamics are two specific projects for which structures determined as part of this Project and results from live cell fluorescence microscopy raise mechanistic questions best answered by frontier methods in cryo-ET. (3) Analysis of transient and multi-state assemblies, including enhancements made possible by the methods developed as part of theme 1 (all four projects). In pursuit of this theme, we will focus especially on the large-scale organization of dynamic structures such as kinetochores, cilia, and transport-vesicle tethering complexes.