Cryogenic electron microscopy single particle analysis (cryoEM-SPA) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) toward a similar resolution, in contrast, is still under active development. Here, we use the archeal chaperonin MmCpn as a model macromolecule to quantitatively investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers, and tilt angle ranges. By delineating the feasibility and experimental factors of attaining near atomic resolution structure with cryoET-STA, especially the effect of electron damage through the tilt series and inelastic scattering at various ice thickness, we encourage a customized tilt series collection strategy for efficient throughput. This study provides a biophysical basis for the application of cryoET-STA (for highly symmetric molecules like MmCpn) toward high resolution and the rationales in using cryoET-STA to achieve an efficient outcome at the desired resolution.

中文翻译：

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近原子分辨率下低分子电子断层扫描伴侣蛋白 MmCpn 亚断层图平均的成本效益分析


低温电子显微镜单颗粒分析 （cryoEM-SPA） 已发展成为以近原子分辨率确定大分子结构的常规方法。相比之下，以相似分辨率为目标的低温电子断层扫描亚断层扫描 （cryoET-STA） 仍在积极开发中。在这里，我们使用 archeal 伴侣 MmCpn 作为模型大分子，在累积电子剂量、冰厚度、亚断层图数和倾斜角范围方面定量研究 cryoET-STA 的分辨率限制因素。通过描述使用 cryoET-STA 获得近原子分辨率结构的可行性和实验因素，特别是电子损伤通过倾斜系列和非弹性散射在不同冰厚度下的影响，我们鼓励定制的倾斜系列收集策略以实现高效通量。本研究为将 cryoET-STA（用于 MmCpn 等高度对称分子）应用于高分辨率提供了生物物理基础，以及使用 cryoET-STA 以所需分辨率获得有效结果的基本原理。