Electron Optic Documents

High resolution structure determination by electron cryo-microscopy (cryoEM) and Single Particle Analysis (SPA) has progressed to the point where structures can be determined routinely to better than 2Å on a 300 kV microscope. Here, we show results from Kato et al. at1 Osaka University from the JEOL CRYO ARM™ 300 installed at SPring8 (Riken, Japan), that was obtained on mouse heavy chain apo-ferritin at 1.5Å resolution. The 3D map shows surprising details in the map reflecting the high resolution quality of the data.

Micro electron diffraction, or microED, is a technique aimed at solving structures of biological macromolecules by electron diffraction. Barn-storming work by the group from Prof. Gonen showed the impressive impact and promise of this technique1. The technique borrows from X-ray crystallography in that precession techniques are used for data collection and that much of the well-established software for solving structures by X-ray crystallography can be used for microED. However, it differs in a fundamental way in that electrons are used, which, owing to the substantially larger scattering cross-section of electrons with biological matter, means much smaller crystals can be used.

CRYO ARM Bibliography

Cryo-EM has seen an enormous increase in capabilities and potential in recent years owing to a number of technological advances, e.g. direct detector devices and improved scope automation. JEOL released two electron cryo-microscopes in 2017 specifically designed for automated and unattended, continuous operation at 200 and 300 kV, the CRYO ARM™ series. A recent update on both type of CRYO ARMs has the potential of increasing the throughput well beyond the current limit of 20,000 images/day, namely north of 50,000 images/day as well as extending the resolution to nearly true atomic resolution, i.e. 1.2Å.

“Visualize the truth” is a hope of researchers who use various measuring equipment. Researchers who use electron microscopes as well have a desire to observe the real structure. But actually, in experiments using electron microscopes, many problems arise: They include damage regions of the specimen when it is cut for the size suited to observation, artifacts due to the staining that is applied to enhance image contrast, deformation caused by substitution of water to resin for withstanding vacuum exposure, and thermal damage to the specimen with electron-beam irradiation. As a result, the visualization of the real structure in the microscope image becomes increasingly difficult. One recommended solution is to cool the specimen, that is, “Cryo” techniques. This “Cryo Note” introduces some of the diversified cryo-techniques. We sincerely hope your challenge to observe the “real structure” will be solved by “Cryo” methods.

Using a multi-hole imaging scheme, researchers have been able to reach a hitherto unprecedented milestone of 20,000 images/day on both a CRYO ARM™ 300 II and a JEM-F200. Given that many structures on EMPIAR have required around 5000 images, essentially 4-5 projects can be accomplished on a daily basis, which opens up new opportunities for routine high resolution structure determination at unprecedented levels.

High resolution structure determination by electron cryo-microscopy (cryoEM) and Single Particle Analysis (SPA) has progressed to the point where structures can routinely be determined to be better than 2Å resolution using either a 200 or a 300 kV microscope. At 1.8Å resolution, details like amino acid isoforms can be distinguished. This application note highlights improved results that were obtained on apoferritin at 1.34Å resolution that hint at new features.

The field of single particle structure analysis (SPA) by cryo-electron microscopy reached new highs with the publication of a 1.19Å structure of apo-ferritin by Maki-Yonekura et al. obtained using a JEOL CRYO ARM™ 300 model 33001. This electron cryo-microscope was specifically designed for highly automated workflows capable of the unattended acquisition thousands of images of vitrified specimens. Workflow support of the JEOL CRYO ARM™ is available for SPA, batch tomography and microED.

Determining the near-atomic resolution structure of a biological macromolecule requires time on a high-end electron cryo-microscope. Depending on the local situation this could mean acquiring images of frozen-hydrated specimens on a JEOL CRYO ARM™ and/or another cryo microscope. To optimize inter-operability between different brands of cryo-microscopes, JEOL have investigated two related aspects: a) the reverse transfer, that is extracting frozen-hydrated specimens from one microscope to be transferred to another one, and b) the usability of a special cartridge designated as AG that are AutoGrid compatible.

Using Minimal Fringe Illumination and Coma-Free Image Shift an unprecedented throughput is possible on a JEOL CRYO ARM™. Given that a typical structure as published on EMPIAR requires 4-5000 images, the potential therefore exists of solving roughly 4-5 structures per day using a JEOL CRYO ARM™.