Biostructural Mechanism Laboratory, RIKEN SPring-8 Center, 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 Jul 1;207(1):40-48. doi: 10.1016/j.jsb.2019.04.011. Epub 2019 Apr 13.
A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.
一种新的 cryo-EM 系统已被用于研究蛋白质结构的单颗粒分析。该系统提供了从冷场发射枪发射的高相干 300kV 电子束的平行照明，并通过柱内能量滤光片和无孔相板提高了图像对比度。它包括用于冷冻样品的自动装载和自动液氮填充，可冷却多个样品。在这项研究中，我们描述了枪和电子束的特性，并证明了该系统适用于单颗粒重构。该系统的性能在两个例子上进行了测试，一个是球形病毒，另一个是脱铁蛋白。还开发了图形用户界面程序来控制和监测系统，以确保正确的照明、较少的椭圆度和稳定的放大倍数，以及闪烁和液氮填充的定时。这些程序对于有效地将该系统应用于单颗粒 cryo-EM 特别有用。
