Tools for High-Throughput High-Resolution Three Dimensional Electron Microscopy

描述(申请人提供)：三维电子显微镜(3DEM)是一套用于确定大型蛋白质和蛋白质复合体结构的技术。已有研究表明，低温电子显微镜(CRYO-EM)有能力确定足够高分辨率的结构，以明确地将侧链放置在EM密度中。尽管低温电子显微镜具有原子分辨的潜力，但要实现低对称性和无对称性络合物的高通量结构测定，仍存在许多障碍。在这里，我们建议为高通量、高分辨率的3DEM开发软件工具。在第一个目标中，我们建议开发用于单粒子3DEM的软件工具，包括用于自动样品筛选的软件、自动数据采集参数优化的软件以及用于处理螺旋样品的基础设施。在第二个目标中，我们建议开发用于层析数据收集和处理的工具，包括双轴倾斜序列的获取，以及用于层析子体处理的开发和基础设施。这项研究将有助于确定与人类疾病相关的蛋白质结构，从而有益于人类健康。

DESCRIPTION (provided by applicant): Three dimensional electron microscopy (3DEM) is a set of techniques for determining the structures of large proteins and protein complexes. It has been shown that cryogenic EM (cryo-EM) has the ability to determine structures to high enough resolution to unambiguously place side chains in the EM density. Though cryo-EM has the potential for atomic resolution, a number of hurdles are present in order to achieve high-throughput structure determination of low-symmetry and no symmetry complexes. Here we propose to develop software tools for high-throughput high-resolution 3DEM. In the first aim, we propose to develop software tools for single particle 3DEM including software for automated specimen screening, automated data acquisition parameter optimization, and an infrastructure for processing helical samples. In the second aim, we propose to develop tools for tomographic data collection and processing including acquisition of dual axis tilt series, and developing and infrastructure for processing tomographic subvolumes. This research will benefit human health by facilitating the determination of protein structures that are relevant for human disease.