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Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo.
PLOS Biology ( IF 7.8 ) Pub Date : 2021-08-26 , DOI: 10.1371/journal.pbio.3001318
Stefano Scaramuzza 1 , Daniel Castaño-Díez 1
Affiliation  

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.

中文翻译:

使用 Dynamo 对病毒样颗粒进行有效子断层图平均的分步指南。

Subtomogram 平均 (STA) 是电子断层扫描中一种强大的图像处理技术,用于确定大分子复合物在其原生环境中的 3D 结构。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 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来通过合理的努力重现结果,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协议,涵盖 STA 中粒子拾取和粒子对齐的所有方面。基于命令行的工作流程完全基于 STA 流行的 Dynamo 软件。在此工作流程中,我们还演示了如何简化和自动化处理管道的大部分以提高吞吐量。该协议面向所有级别的用户。它可以用于培训目的,也可以作为基础,通过修改和扩展给定的管道,利用 Dynamo 的灵活性来设计用户特定的项目。使用来自未成熟 HIV-1 病毒样颗粒 (VLP) 的电子显微镜公共图像存档 (EMPIAR) 数据库条目 10164 成功验证了该协议,这些颗粒描述了电子断层扫描中常见的几何形状。该协议面向所有级别的用户。它可以用于培训目的,也可以作为基础,通过修改和扩展给定的管道,利用 Dynamo 的灵活性来设计用户特定的项目。使用来自未成熟 HIV-1 病毒样颗粒 (VLP) 的电子显微镜公共图像存档 (EMPIAR) 数据库条目 10164 成功验证了该协议,这些颗粒描述了电子断层扫描中常见的几何形状。该协议面向所有级别的用户。它可以用于培训目的,也可以作为基础,通过修改和扩展给定的管道,利用 Dynamo 的灵活性来设计用户特定的项目。使用来自未成熟 HIV-1 病毒样颗粒 (VLP) 的电子显微镜公共图像存档 (EMPIAR) 数据库条目 10164 成功验证了该协议,这些颗粒描述了电子断层扫描中常见的几何形状。
更新日期:2021-08-26
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