Max Planck Institute of Biophysics, Frankfurt on Main, Germany.
Buchmann Institute for Molecular Life Sciences, Goethe University of Frankfurt on Main, Frankfurt, Germany.
Nat Commun. 2023 Oct 17;14(1):6543. doi: 10.1038/s41467-023-42085-w.
Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR-a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent ("white box") approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.
天然状态下的大分子结构为其功能提供了独特而明确的见解。冷冻电子断层扫描结合亚断层平均技术证明了在原位以 3 埃分辨率解决此类结构的能力，对于某些大分子来说，分辨率在 3 埃范围内。为了能够适用于在细胞中以有限量观察到的大多数大分子的结构测定，必须以高通量的方式处理断层扫描数据。在这里，我们提出了 TomoBEAR，这是一个用于亚断层平均的冷冻电子断层扫描数据的简化处理的模块化可配置工作流引擎。TomoBEAR 将常用的冷冻电镜软件包与合理的预设相结合，为数据管理和处理提供了一种透明的（“白盒”）方法。我们将 TomoBEAR 应用于两个纯化的大分子靶标数据集、一个膜中的离子通道 RyR1 以及等离子体 FIB 铣削薄片的断层扫描图，并证明了生成高分辨率结构的能力。TomoBEAR 加快了数据处理速度，最大限度地减少了人工干预，并将有助于加速冷冻电子断层扫描的原位结构生物学的采用。源代码和文档均可免费获取。
