Automated cryoEM: Integrating tilted data collection and analysis

描述（由申请人提供）：在之前的资助期内成功开发了螺旋标本的自动冷冻显微镜管道，随后建立了国家自动分子显微镜资源，为当前的应用奠定了基础。在自动冷冻电镜中，一个关键的缺失组件是确定未知大分子复合物的明确初始模型的手段，以及分析样品中构象异质性程度的基础设施。确定各种构象状态下大分子复合物结构的能力是低温电子显微镜的下一个重大挑战，这代表了解决有趣的生物学问题的独特机会。我们对这一挑战的贡献将是设计、实施和测试一个单颗粒结构分析系统，该系统使用实验而不是计算策略来计算初始模型，确定样品中构象和取向变化的程度，并在细化过程中拒绝不完美的颗粒。为了实现这一目标，我们将开发程序，以确定使用层析成像和随机锥形数据采集方法的初始模型集，以及用于倾斜图像对的大规模数据收集的程序。数据获取和分析程序将由一个基于网络的图形用户界面支持，该界面将与一个关系数据库紧密结合。这项工作代表了国家自动分子显微镜资源（NRAMM）目前正在进行的努力的重要延伸。

DESCRIPTION (provided by applicant): The successful development of an automated cryoEM pipeline for helical specimens in the previous grant period and the subsequent establishment of the National Resource for Automated Molecular Microscopy sets the stage for this current application. One of the critical missing components in automated cryoEM is the means to determine unambiguous initial models for an unknown macromolecular complex and the infrastructure to analyze the degree of conformational heterogeneity in the specimen. The ability to determine the structure of macromolecular complexes in a variety of conformational states is the next big challenge for cryoEM, representing a unique opportunity to address interesting biological questions. Our contribution to this challenge will be to design, implement and test a system for single particle structural analysis that uses experimental, rather than computational, strategies for calculating an initial model, determining the degree of conformational and orientational variability in a specimen, and rejecting imperfect particles during refinement. To achieve this we will develop procedures to determine sets of initial models using both tomographic and random conical data acquisition methods, and procedures for large scale data collection of tilted pairs of images. The data acquisition and analysis procedures will be supported by a web based graphical user interface that will be tightly integrated with a relational database. This work represents a significant extension of the current efforts underway at the National Resource for Automated Molecular Microscopy (NRAMM).