Application of a Helium-Cooled Cryo-Electron Microscope for Single Particle Analysis

Single particle analysis can determine three-dimensional structures of biological molecules at high resolution without the crystals. The principle obstacle for high-resolution structural analysis in electron microscopy is irradiation damage of the biological specimen, and cooling the specimen to cryogenic temperatures can reduce the effects of irradiation damage. To achieve higher resolution structures by reducing the irradiation damage, even in single particle analysis, a new cryo-electron microscope with a liquid helium-cooled stage for single particle analysis was developed, and we utilized it in two studies using single particle analysis. For the application, we slightly modified the sample preparation technique developed for the high-resolution image collection from the tubular crystals, using the liquid helium-cooled stage. The first application was to determine the structure of the GroEL-GroES-substrate complex from Thermus thermophilus.
We observed a density corresponding to the folding substrates inside the complex. The second application was the threedimensional reconstruction from tubular crystals of a membrane protein, human erythrocyte band 3. Iterative helical real-space reconstruction,which is a modification of single particle analysis for tubes with helical symmetry, was used for this purpose. These two examples clearly show that the three-dimensional structures of a broad range of targets can be analyzed by single particle analysis.