Structure determination of frozen, hydrated, crystalline biological specimens

Low temperature transmission electron microscopy can be used to study the structure of biological materials in the hydrated state. Spatial averaging techniques are necessary to overcome the radiation damage problems, and for this reason the techniques described are most applicable to crystalline objects. However, with thin, crystalline biological specimens there are no difficulties with preserving periodicity during the freezing process. Improved specimen preparation methods are described which achieve the production of very thin aqueous films with the hydrated specimen embedded in them. Defocused bright field images of frozen, hydrated protein crystals possess a surprisingly high contrast, presumably due to the difference in density of the protein and the aqueous phase. The techniques described have been used to study the crystal structure of hydrated catalase and the outermost cell wall of Spirillum serpens.