High resolution cryo-electron microscopy for biological macromolecules.

The irradiation damage of the crystals of proteins as well as nucleic acid is reduced almost exponentially even below the liquid nitrogen temperature down to 8K provided that the specimen area under illumination is cooled down. A cryo-stage cooled by superfluid helium is designed for the high resolution electron microscope based on the idea of the helium evaporation refrigerator with a capillary which was developed by Delong et al. We also developed a new top-entry-type cryo-transfer system for this superfluid cryo-electron microscope. The images of chlorinated Cu-phthalocyanine are taken to estimate the resolution attainable by this cryo-stage at 1.5 K with accelerating voltage of 400 kV. The optical diffraction confirms the resolution of 0.26 nm. The complexes of rec A proteins and DNA molecule are clearly visible in vitreous ice. A new membrane structure of the influenza virus is observed in the case of influenza A virus.