Department of Biochemistry, Duke University School of Medicine, Durham, NC 27708, USA.
Department of Computer Science, Duke University, Durham, NC 27708, USA.
Acta Crystallogr D Struct Biol. 2022 Jul 1;78(Pt 7):817-824. doi: 10.1107/S2059798322005010. Epub 2022 Jun 7.
Tomographic reconstruction of frozen-hydrated specimens followed by extraction and averaging of sub-tomograms has successfully been used to determine the structure of macromolecules in their native environment at resolutions that are high enough to reveal molecular level interactions. The low throughput characteristic of tomographic data acquisition combined with the complex data-analysis pipeline that is required to obtain high-resolution maps, however, has limited the applicability of this technique to favorable samples or to resolutions that are too low to provide useful mechanistic information. Recently, beam image-shift electron cryo-tomography (BISECT), a strategy to significantly accelerate the acquisition of tilt series without sacrificing image quality, was introduced. The ability to produce thousands of high-quality tilt series during a single microscope session, however, introduces significant bottlenecks in the downstream data analysis, which has so far relied on specialized pipelines. Here, recent advances in accurate estimation of the contrast transfer function and self-tuning exposure-weighting routines that contribute to improving the resolution and streamlining the structure-determination process using sub-volume averaging are reviewed. Ultimately, the combination of automated data-driven techniques for image analysis together with high-throughput strategies for tilt-series acquisition will pave the way for tomography to become the technique of choice for in situ structure determination.
冷冻水合标本的断层重建，接着是亚断层的提取和平均，已成功用于确定其天然环境中大分子的结构，分辨率高到足以揭示分子水平的相互作用。然而，断层数据采集的低通量特性，以及获得高分辨率图谱所需的复杂数据分析管道，限制了该技术在有利样本或分辨率方面的适用性，这些分辨率太低，无法提供有用的机械信息。最近，引入了一种显著加速倾斜系列采集而不牺牲图像质量的策略——光束图像移动电子冷冻断层扫描（BISECT）。然而，在单个显微镜会议期间能够生成数千个高质量的倾斜系列，这在下游数据分析中引入了显著的瓶颈，迄今为止，该分析依赖于专门的管道。在这里，对对比度传递函数的精确估计的最新进展，以及自我调整曝光加权例程的综述，这些进展有助于提高分辨率和简化使用子体积平均法进行结构确定的过程。最终，自动化的数据驱动技术与高通量倾斜系列采集策略的结合，将为断层扫描成为原位结构确定的首选技术铺平道路。
