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We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope—the first machine of this model—and a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly coherent 300-kV electron beam to a sample from a cold-field emission gun and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress and future plan for running such cryo-EM machines in RIKEN SPring-8 Center. [ABSTRACT FROM AUTHOR]

Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera.

Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Takaba, Maki-Yonekura, Inoue, Hasegawa and Yonekura.)