Automated, optimized, intelligent data collection for cryo-EM

项目摘要 冷冻电子显微镜(Cryo-EM)现在是一种广泛建立和不可或缺的方法来确定 生物医学上重要分子的高分辨率结构。鉴于数以千计的图像，通常是获得的 在过去的几天里，都需要获得这样的结构，自动化软件起到了至关重要的作用 在科学界大规模采用这种方法的过程中发挥了重要作用。就在过去的五年里，冷冻EM 彻底改变了我们对整个生物系统的理解，并在2020年提供了第一个分子 SARS-CoV-2与中和抗体相互作用的描述。冷冻EM的广泛采用 最近促使美国国立卫生研究院通过变革性的高分辨率冷冻技术投资了三个国家中心- 电子显微镜计划，为世界各地的生物学家提供免费的高端电子显微镜 国家。冷冻-EM的受欢迎程度的指数增长导致了惊人的数量的发展 在样品制备方法和图像处理算法中，这些方法和算法改进了可实现的 单粒子重建的分辨率。然而，在优化方面进展相对较小。 正在收集的低温电磁数据的质量。开创性的软件包Leginon和Appion 分别演示了自动数据采集和实时处理的能力，并有 现在有许多用于自动数据采集和实时处理的程序。尽管取得了进展，但 自动化，以最佳方式从EM样本中提取最高质量的数据仍然需要人工参与 一个专业的电子显微镜专家。运行适当的映像需要用户干预和专业知识 分析、解释结果，并就处理结果与正在进行的结果之间的关系做出明智的决策 数据收集。然而，即使是专家也必须满足于这样一个事实，即“最佳网格区域”差别很大。 从一个样本到另一个样本，而且还没有既定的工具来自动和快速地评估 横跨EM网格的各种微环境的样本。考虑到不断增加的合并 为了将冷冻-EM纳入实验室的研究计划，必须简化数据收集和处理，以 满足结构社区日益增长的需求。我们计划开发第二代 Leginon/Appion软件包“Magellon”，以克服现有的瓶颈，并提供一条途径 全自动数据采集，在数据采集过程中不需要用户输入。重要的是，这 软件将支持计算基础设施，以实现实时图像处理结果通知 并修改正在进行的数据收集制度，了解在将产生 最高分辨率的结构。我们将开发和整合新的快速图像评估程序，同时还 提供应用程序编程接口，以支持从 社区中的开发人员。此外，麦哲伦将实现直接、无缝的数据导入和导出 从其数据库中获取数据，以便在任何区域或国家低温电磁中心进行远程数据采集。

Project Summary Cryo-electron microscopy (cryo-EM) is now a widely established and indispensable method for determining the high-resolution structures of biomedically important molecules. Given that thousands of images, often acquired over the course of several days, are required to obtain such structures, automation software has played a critical role in the large-scale adoption of this method by the scientific community. In just the past five years, cryo-EM has revolutionized our understanding of entire biological systems, and in 2020 provided the first molecular descriptions of SARS-CoV-2 interaction with neutralizing antibodies. The widespread adoption of cryo-EM recently prompted the NIH to invest in three National Centers through the Transformative High Resolution Cryo- Electron Microscopy Program, providing free, high-end electron microscope access to biologists across the country. The exponential increase in the popularity of cryo-EM has led to an astonishing number of developments in sample preparation methodologies and image processing algorithms, which have improved attainable resolution of single particle reconstructions. However, comparatively little progress has been made in optimizing the quality of the cryo-EM data being collected. The pioneering software packages Leginon and Appion demonstrated the power of automated data acquisition and real-time processing (respectively), and there are now numerous programs for automated data acquisition and real-time processing. Despite advances in automation, optimally extracting the highest quality data from an EM sample still requires manual involvement of an expert electron microscopist. User intervention and expertise is necessary to run the appropriate image analyses, interpret the results, and make informed decisions on how the processed results relate to the ongoing data collection. However, even experts must content with the fact that the “best grid regions” differ drastically from sample to sample, and there are no established tools for automatically and quickly assessing the quality of the specimen across the various microenvironments of an EM grid. Given the ever-increasing incorporation of cryo-EM into labs’ research programs, it is imperative that data collection and processing be streamlined to match the growing needs of the structural community. We propose to develop a second generation Leginon/Appion software package, “Magellon”, to overcome existing bottlenecks and provide an avenue toward fully automated data acquisition that bypasses need for user input during data collection. Importantly, this software will support the computational infrastructure to enable real-time image processing results to inform on and modify the ongoing data collection regime by learning where to acquire images in regions that will yield the highest resolution structures. We will develop and incorporate new, fast image assessment routines, while also providing an application programming interface to enable the incorporation of extensions and plugins from developers in the community. Further, Magellon will enable straightforward, seamless import and export of data from its database to accommodate remote data acquisition at any of the regional or national cryo-EM centers.