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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.

• 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]

• 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]

• Tools for manipulating cryo-EM grids are designed and named CryoGridTools. • Each tool is easily made using a wide-use 3D printer and polished under acetone gas. • The grid case container is light and stackable in a standard conical tube. • The supporting tools facilitate handling of grid cases under liquid nitrogen. • CryoGridTools organizes many grids and the blueprints are available from our website. Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases. These tools are made of a plastic fiber using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas. A grid case stacker organizes many frozen-hydrated cryo-EM grids and the stackers can be piled up inside a standard 50 mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. Blueprints of the tools named CryoGridTools are available from a GitHub site. [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., 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.)