Biostructural Mechanism Laboratory, RIKEN, SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; Advanced Electron Microscope Development Unit, RIKEN-JEOL Collaboration Center, RIKEN Baton Zone Program, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
J Struct Biol. 2019 May 1;206(2):243-253. doi: 10.1016/j.jsb.2019.03.009. Epub 2019 Mar 27.
A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.
一种新的 cryo-EM 系统已被开发并应用于蛋白质电子 3D 晶体学。该系统提供对相干 300kV 电子束的平行照明，通过柱内能量过滤器对样品进行能量损失电子过滤，并允许在快速相机上进行旋转数据采集。它还具有用于冷冻样品加载和自动液氮填充的电动装置，以冷却多个样品。为了方便使用，我们开发了用于高效操作和准确结构分析的 GUI 程序。在这里，我们报告了该系统的性能以及蛋白质复合物过氧化氢酶和膜蛋白复合物 ExbBD 的薄 3D 晶体的初步结果。与不进行能量过滤的 200kV 收集相比，该方法显著提高了数据质量，我们称之为 eEFD（3D 晶体的电子能量过滤衍射）。关键进展包括显微镜的精确控制和镜头波动的记录，程序对其进行处理和响应。我们还讨论了在电子 3D 晶体学中使用更高能量电子和能量损失电子过滤的优点。
