V-ATPase is a rotary molecular motor which couples ATP synthesis/hydrolysis in the soluble domain with proton flow in the membrane domain by a rotation of the central rotor complex. Here, we present three rotational states of V-ATPase from the bacterium Thermus thermophilus by single particle analysis using cryo-EM. By using a combination of masked classification/refinement and focused classification techniques, the resolutions of these EM maps were improved. These EM maps provide the first detailed insight into the contact surface between each subunit, and the assignment of the movement of each subunit during rotation. In this article, I would like to report the results of structural analysis of V-ATPase and introduce the actual situations of sample preparation and analytical methods for single particle analysis.
Recently, electron cryo-microscopy single particle analysis (cryo-EM SPA) has been developed dramatically and can be used for drug design because of the greatly improved resolution to near-atomic level. Nowadays, based on the atomic structures of drugs within the target protein molecules, the drugs can be designed or modified more efficiently, which is called as structure-based drug design (SBDD). We have constructed a cryo-EM system for supporting collaborations for drug discovery and life sciences, mainly supported by and used for national platform projects of BINDS and PDIS, AMED. For example, structures of a virus-like particle of PfV (Pyrococcus furiosus virus-like particle) of 30 nm in diameter have been determined at near-atomic resolutions (3.3 Å) by both conventional cryo-EM (CTEM) and Volta phase contrast cryo-EM (VPC-TEM) SPA methods within a week. In addition to the high-throughput structure analysis, we succeeded high-resolution cryo-EM SPA at 2.3 Å resolution with an advanced direct electron detecting camera, Falcon 3EC in an electron counting mode. Here, we introduce our cryo-EM system for the high-throughput and high-resolution single particle analyses.
The structural analysis of a biological samples are carried out by X-ray crystallography, NMR and electon cryomicroscopy(CryoEM). Only until a few years ago, the resolution of the structural analysis by CryoEM were not so high than the others. But by development of direct electron detection camera, the resolution by CryoEM was dramatically improved. Therefore we have been developing new cryoEM with JEOL to high resolution analysis more easily. This high-end CryoEM equipped with an automatic sample exchange device that can automatically, cryo pole piece specialized for single particle image analysis and Ω type energy filter for contrast improvement.To evaluate the resolution by single particle image analysis, β-galactosidase was used as a test sample. The 2,500 images were automatically collected by “JADAS” produced by JEOL. Finally 88,000 particle images were used for single particle image analysis by Relion. As a result, the resolution was achieved to 2.6 Å, the density map of some aromatic ring had a hole, and Mg ion density was clearly observed. This resolution is one of highest one in the world at 200kV CryoEM. We demonstrated that the Cryo ARM 200 is possible to sufficiently analyze high resolution analysis.
Cryo-electron tomography is a powerful method to visualize the three-dimensional structures of macromolecular complexes and cellular organelles in situ. Compared with the single particle analysis, however, cryo-electron tomography appears to be relatively unpopular probably due to the seemingly complicated methodology. In this review, I will give an overview of the practical aspects of the cryo-electron tomography as well as the recent progress in this field of research.
Bone is one of the hard tissues in living vertebrates. Homeostasis of the skeletal system, bone morphology and mineral metabolism, which is called bone metabolism, is highly regulated through balancing between bone resorption and bone formation. Bone metabolism is tightly associated with histological morphology of the bone. Therefore, microscopic analysis has been a gold-standard approach for understanding bone pathophysiology. This short technical review scopes the application of current microscopic innovations including super-resolution microscopy (especially, structured illumination microscope) to the bone biomedicine by introducing our recent works.
Magnetic skyrmions are magnetic vortex-like topological particles excited in magnetic materials with external magnetic fields. In the skyrmion, electron-spins swirl from the north-pole in the core to the south-pole in the peripheral of the skyrmion. Nanometric skyrmions as information carriers are promising for applications to the high-density and low-power-consumption magnetic memory owing to their topological novelty. The real-space observation of skyrmion is more essential to understand the skyrmion and thereby control it. We report here the Lorentz transmission electron microscopy (Lorentz TEM) studies on various magnetic skyrmions under external magnetic fields in helimagnets/uniaxial ferromagnets with non-centrosymmetric/centrosymmetric structures.
Based on the first lecture on ‘Electron sources and guns 1,’ which discussed the optical/physical properties of electron sources, this second article deals with two fundamental designing technologies of electron guns. The one is the vacuum technology. The operation principles of cold field emitters (CFEs) are described from the viewpoint of vacuum science. The advantages of extremely high vacuum (EHV) over conventional ultra high vacuum (UHV) are explained in terms of the probe current stability and the high current capability. The second is the optical designing technique to achieve a high probe current. The point cathode optics require the consideration of ‘electron gun aberrations’ since its virtual source is very small and the angular current intensity low. The introduction of an optical parameter ‘electron gun focal length’ enables quantitative evaluation of gun aberration influences and the optimization of lens operation conditions.
Nanoscale imaging of the biological specimens and/or organic materials under water condition provides valuable insight to the analysis of the biological mechanisms. In particular, scanning electron microscopy (SEM) has widely been used to analyse the organic nano-particles, bacterial and protein structures. However, SEM observations of these specimens under high vacuum condition require specific sample preparation protocols to enhance contrast and to avoid electrical radiation damage. We recently developed a new imaging technology called a scanning-electron assisted dielectric-impedance microscopy (SE-ADM) system based on SEM. Our system provides high-contrast imaging and low-radiation damage for the observation of the intact biological specimens in water using an atmospheric sample holder. Furthermore, it can be used for diverse liquid samples across a broad range of scientific fields, such as nanoparticles, organic materials and catalytic materials.
In scanning transmission electron microscopy (STEM), one can obtain a variety of STEM images such as bright-field (BF), annular dark-field (ADF) and differential phase contrast (DPC) images with variously shaped detectors. However, the information in a diffraction patterns on the detector plane is not fully utilized, when we use conventional detectors, as they integrate the intensity over the scintillator. Meanwhile, direct electron detectors whose frame rate and number of pixels are thousands fps and more than several ten thousand pixels, have recently been commercialized and used in STEM. Such detectors, when used for recording the diffraction patterns for STEM probe positions, are called pixelated STEM detectors. With the obtained 4-dimensional dataset, a variety of STEM images can be synthesized in a post or real time processing. We have developed a fast pixelated STEM detector and integrated it into our electron microscopes. Here, we report the hardware and its application data taken with the detector.