Automated cryoEM: Integrating tilted data collection and analysis

描述(由申请人提供)：在前一批赠款期间，成功开发了用于螺旋样本的自动低温EM管道，随后建立了国家自动分子显微镜资源，为目前的申请奠定了基础。自动低温电子显微镜中缺少的关键部件之一是确定未知大分子复合体的明确初始模型的方法，以及分析样品构象异质性程度的基础设施。确定各种构象状态下的大分子复合体结构的能力是CryoEM面临的下一个重大挑战，这代表着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、实现和测试一个用于单粒子结构分析的系统，该系统使用实验而不是计算的策略来计算初始模型，确定样本中构象和取向的可变性程度，并在精化过程中排除不完美的粒子。为了实现这一点，我们将制定程序，使用层析和随机圆锥数据采集方法来确定初始模型集，并制定大规模倾斜图像对的数据收集程序。数据采集和分析程序将得到一个基于网络的图形用户界面的支持，该界面将与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)目前正在进行的工作的重要扩展。

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).