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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 semi-automated protocol has been developed for rotational data collection of electron diffraction patterns. • The protocol was tested on a general-purpose JEM-2100 and a high-end CRYO ARM 300 electron microscopes. • Combined use of SerialEM and ParallEM proved to be efficient and accurate in data collection of large-scale rotational series. • The development also supports periodic flashing and pausing of data collection during day-long recording with a cold field-emission beam. A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12–20 rotational series from ~−68° to ~68° at a rotation speed of 1°/s in one hour without human supervision. [ABSTRACT FROM AUTHOR]