Key Laboratory of Protein Sciences ( Tsinghua University ), Ministry of Education, Beijing 100084 , China.
School of Life Sciences , Tsinghua University , Beijing 100084 , China.
Anal Chem. 2019 Sep 3;91(17):10996-11003. doi: 10.1021/acs.analchem.9b01162. Epub 2019 Aug 15.
Microcrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lack of automated data collection methods. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with a single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ∼0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED and demonstrates the capability of the low-end 120 kV microscope with a CCD camera in solving ultrahigh resolution structures of both organic compounds and biological macromolecules.
微晶体电子衍射（MicroED）正在成为确定生物大分子和小分子有机化合物晶体结构的有力工具。然而，该技术的广泛应用仍然受到耐辐射电影模式相机的特殊要求和缺乏自动化数据采集方法的限制。在此，我们开发了一种相机同步方案，以最小化硬件要求并实现使用带有单帧 CCD 相机的传统电子冷冻显微镜，这不仅确保了超高分辨率衍射数据的获取，而且在实际中也降低了成本。该方法可用于超高分辨率（高达约 0.60Å）的肽和小分子有机化合物的结构测定，并能明确分配几乎所有的氢原子。本工作为 MicroED 的常规结构测定提供了一种广泛适用的解决方案，并展示了带有 CCD 相机的低端 120kV 显微镜在解决有机化合物和生物大分子的超高分辨率结构方面的能力。
