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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]

• A cryo-EM with a cold-field emission gun (CFEG) was tested for single particle analysis. • GUI programs were developed for an efficient and accurate data collection. • Imaging with CFEG is beneficial to obtaining high-resolution structures by single-particle cryo-EM. 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. [ABSTRACT FROM AUTHOR]

• A new cryo-EM system was developed and tested for electron 3D crystallography. • GUI programs was developed for an efficient and accurate structure analysis. • Data quality is remarkably improved by eEFD with this system. • Higher-energy electrons and energy filtration are highly beneficial. 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. [ABSTRACT FROM AUTHOR]