Atomic Resolution in Biological Electron Microscopy

项目描述（由申请人提供）：本项目目前已进入第十个年头，主要目标是开发电子低温显微镜（cryoEM）的实验和计算工具，以扩大生物应用范围。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推向成为高分辨率工具。在过去的资助期内，几项进展之一是达到了几种病毒结构的近原子分辨率（<4A），实现了亨德森15年前的一个猜想，即通过低温电子显微镜在这种细节水平上对生物组装进行成像是可能的。我们为即将到来的项目提出三个主要主题。(1)持续的方法开发。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装的图像（Grigorieff, Harrison）；我们将改进样品制备的均匀性和同质性，在上一个拨款期间建立亲和网格的发展（Walz）；我们将探索减少梁引起的运动的方法（Grigorieff）；提高细胞成像的分辨率（Nicastro）。(2)电子冷冻层析成像（cryo-ET）是通过光学显微镜和活细胞成像实现近原子分辨率结构可视化和细胞内动力学研究的桥梁（Nicastro, Harrison, Grigorieff）。轮状病毒侵入和网格蛋白包被动力学是两个特定的项目，其结构作为本项目的一部分确定，并从现场得到结果

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.