High-resolution structure determination using high-throughput electron cryo-tomography.
High-resolution structure determination using high-throughput electron cryo-tomography.
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高通量电子冷冻层析成像的高分辨率结构测定。
DOI：
10.1107/s2059798322005010
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发表时间：
2022-07-01
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在这篇文章中，它表明，高通量的战略相结合的断层扫描数据采集与无监督的图像分析技术提供了基础，用于研究在体外重组的分子组装体和原位结构测定技术的高分辨率的战略之间的分辨率差距关闭。断层重建的冷冻水合标本，然后提取和平均的子断层图像已成功地用于确定在其原生环境中的大分子的结构，在分辨率高到足以揭示分子水平的相互作用。然而，层析成像数据采集的低吞吐量特性与获得高分辨率图所需的复杂数据分析管道相结合，限制了该技术对有利样品或分辨率太低而不能提供有用的机械信息的适用性。最近，引入了束图像移位电子冷冻断层扫描（BISECT），这是一种在不牺牲图像质量的情况下显著加速倾斜系列采集的策略。然而，在单个显微镜会话期间产生数千个高质量倾斜系列的能力在下游数据分析中引入了重大瓶颈，迄今为止，下游数据分析一直依赖于专门的管道。在这里，最近的进展，准确估计的对比度传递函数和自调整的分辨率和简化的结构确定过程中，使用子体积平均有助于提高权重的例程进行审查。最终，自动数据驱动的图像分析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。
In this article, it is shown that high-throughput strategies for tomographic data acquisition combined with unsupervised techniques for image analysis provide the foundation for closing the resolution gap between the high-resolution strategies used to study molecular assemblies reconstituted in vitro and techniques for in situ structure determination. 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.