BioEM Lab, Biozentrum, University of Basel, Basel, Switzerland.
PLoS Biol. 2021 Aug 26;19(8):e3001318. doi: 10.1371/journal.pbio.3001318. eCollection 2021 Aug.
Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line-based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography.
子断层平均（Subtomogram averaging，STA）是一种用于确定天然环境中大分子复合物 3D 结构的电子断层摄影术中强大的图像处理技术。它是一种快速发展的技术，在结构生物学中越来越重要。STA 的计算方面非常复杂，取决于大量变量。我们注意到缺乏关于 STA 处理的详细指南。此外，该领域的当前出版物通常缺乏足够实用的文档，无法在合理的努力下重现结果，这对于科学界的发展是必要的。因此，我们提供了一个完整、详细且完全可重复的处理方案，涵盖了 STA 中粒子挑选和粒子对准的所有方面。基于命令行的工作流程完全基于流行的 STA 软件 Dynamo。在这个工作流程中，我们还展示了如何通过利用 Dynamo 的灵活性，通过修改和扩展给定的管道，对处理管道的大部分进行简化和自动化，以提高吞吐量。该方案面向各级用户。它可以用于培训目的，也可以通过利用 Dynamo 的灵活性，通过修改和扩展给定的管道，为设计特定用户的项目提供基础。该方案使用来自不成熟 HIV-1 病毒样颗粒（VLPs）的 Electron Microscopy Public Image Archive（EMPIAR）数据库条目 10164 成功验证，该条目描述了电子断层摄影术经常看到的一种几何形状。
