The three-dimensional structure of biological macromolecules and their complexes is the fundamental information not only for life sciences but also for medical sciences and drug design. Electron cryomicroscopy has become an extremely powerful tool for high-resolution structural analysis of biological macromolecules, not any more supplementary to X-ray crystallography and NMR that have been used as the basic technique in structural biology. By the development of hardware and software, such as transmission electron cryomicroscopes with highly stable and controllable electron optics, cold field emission gun and energy filter, CMOS-based direct electron detectors with high frame rate and high sensitivity, high-speed computers and software programs for image analysis, electron cryomicroscopy now allows atomic-resolution structure determination of biological macromolecules within a few days even from a drop of solution sample with an amount as small as a few μg. How can the structures of macromolecules be imaged and analyzed at atomic level resolution in their native states despite their high sensitivity to radiation damage? We describe recent advances and future prospect of electron cryomicroscopy for structural life sciences.

In recent years, the resolution of protein structural analysis by cryo-EM was dramatically improved, due to the using a new type of camera that can detect electrons directly named direct electron detection camera, and it is now possible to construct an atomic model using only cryo-EM structural analysis. It is now the first choice for structural analysis of protein molecules with molecular weights exceeding 300 kDa, and has now established itself as a powerful tool for structural analysis. In order to make this technology available to as many researchers as possible, cryo-EM facilities for shared use have been established around the world. In Japan, 19 cryo-EMs at 9 facilities are in operation, and anyone can easily receive support for their use through the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS). This review introduce of the flow of establishment of joint-use facilities in Japan, and discusses the current issues, and future prospects.

About 10 years have passed since the resolution revolution in cryo-EM single-particle analysis (SPA) occurred. During this period, state-of-the-art cryo-EM equipment was rapidly introduced mainly in scientifically advanced countries. Atomic resolution in cryo-EM SPA will be achieved in 2020, and the ripple effect will be even greater. Under these situations, the National Institutes of Natural Sciences (NINS) have introduced a 300 kV cryo-EM, which has recorded the world’s highest performance, and has started joint use by industry, academia, and research institutes in Japan and overseas from FY2022. We will also introduce the latest cryo-FIB-SEM as a cryotomography sample preparation device, and start research aimed at structural analysis of proteins in cells. By combining these with the biomolecular dynamics and function analysis system has been operated at our institutes, we will provide unique research support that seamlessly connects the protein structure and dynamic function. This paper introduces the details and the current status.

Electron 3D crystallography, which is now considered as one of the major cryo-EM modalities, can determine 3D structures from undersized crystals. It can even reveal sub-atomic resolution structures of various samples including organic compounds, which are not compatible with single particle cryo-EM and cryo-electron tomography. Thus, its application is not limited to life science but extended to synthetic organic chemistry, pharmaceutical and material sciences, and related areas. We have been involved in development of this technique since its early stage, and now rapid structure determination is routinely done by AI data collection and automated data processing from small crystals in organic solvent, water solution, and powders. This review covers the background of this technique and our recent results on a new double helix structure built by self-assembly of nanographene, thin crystals of an organic semiconductor, and fibrous crystals of polypeptides related to a neurological disease, amyotrophic lateral sclerosis (ALS). The structure of the organic semiconductor includes disorder regions, which may be useful for design of new soft devices. The polypeptide structures of the wild-type and a toxic mutant may suggest how amyloid fibers aggregate. We also discuss the limitation and merits of electron diffraction in comparison with XFEL crystallography.

Ribosomes decode genetic information encoded mRNA and synthesize corresponding proteins, which exert various functions in biological activities. This macromolecular assembly is more than 25 nm in diameter, and its core is mainly comprised of RNA moiety covered with many proteins. The history of the visualization of ribosomes has been with electron microscopy. The observation of ribosomes started in the early 1960s using negative staining microscopy, followed by cryo-electron microscopy revealed its high-resolution structures and dynamics. Now, cryo-EM has become an essential tool for understanding the basic principle of translation. This review overviews the history and recent advancements in the structural analysis of ribosomes.

Atomic structure observation of magnetic materials has been extremely challenging with conventional high-resolution electron microscopes because the sample must be placed in a intense magnetic field of 2 T or higher during observation. To overcome this limitation, we have developed an electron microscope that enables atomic-resolution with a magnetic field-free environment around the sample. This electron microscope facilitates atomic structure analysis of magnetic materials and is expected to realize the high resolution observation of magnetic structures in materials using differential phase contrast methods. This paper shows the development of the atomic-resolution magnetic field-free electron microscope and the real-space observation of magnetic fields of antiferromagnetism of α-hematite at atomic resolution using this electron microscope.

Cryo-SEM can visualize ultra-structures of hydrated or liquid specimens such as tissues, cells, gels, emulsions, and slurries without dehydration. General cryo-SEM is equipped with a cryo-stage and a cold trap on a room temperature SEM. For cryo-SEM observation it is important to freeze hydrated specimens in an amorphous state. After amorphous freezing, the specimen should be cross-sectioned to exposure the internal structure. Depending on the observation targets, we should choose a cross-sectioning method between freeze-fracturing, cryo-ultra-thin-sectioning, or ion milling. In many cases we can observe the cross-sectioned specimens without any coating even if the specimen is non-conductive. If composite images are not observed on the flat sectioned surface, the specimen should be heated to at around –100°C to sublimate the surface amorphous ice and exposure the observation targets above the cross-sectioned surface. If observation is difficult by the sample charging, conductive metal or carbon coating could help clear imaging. Cryo-SEM is a powerful tool to analyze sub-micrometer structures and their distribution or localization in hydrated or liquid specimens, since SEM allows observation extending from low magnification to high magnification.