Potential for High‐Resolution Electron Crystallography at Intermediate High Voltage a

Electron crystallography is a unique technique for three-dimensional structure determination of macromolecules. Differing from X ray crystallography, this technique uses thin, small crystals and can retrieve the structural phases directly from electron images.’ Henderson and Unwin demonstrated the usefulness of this technique with their determination of the structure of the purple membrane. They preserved the two-dimensional crystals of bacteriorhodopsin by embedding them in a thin layer of glucose, and reduced the electron radiation damage of the specimen by using a very low electron exposure.’ Computer processing techniques were subsequently used to obtain the three-dimensional structure of bacteriorhodopsin to 7-A resolution. The seven rods of mass density in their reconstruction were interpreted as seven he lice^.^ Since then, an increasing number of two-dimensional crystals, particularly of membrane proteins, have been studied by the electron crystallographic technique. However, most of the three-dimensional structures have been limited to about 20-A resolution primarily due to the poor crystallinity of these specimens or to specimen preparation techniques4 We have made a number of thin crystals of soluble proteins that are well suited for high-resolution electron crystallographic analy~is .~” In order to record high-resolution electron images the specimens must be kept a t low temperature to minimize radiation damage. The electron microscope must therefore be equipped with a mechanically stable cold stage. Images of the crotoxin complex, purple membrane, and paraffin crystals have been recorded to 3.5 A or better in cryomicroscopes.”’ However, because of the low contrast in the structure factors for reflections beyond 7 A, it is difficult to detect them in the optical diffraction patterns of the images.” Hayward and Stroud enhanced the detectability of the high-resolution structure factors by superimposing small image patches of purple membrane, and justified the validity of the high-resolution structural data by evaluating the probability distribution function of the phases and their figures of merit.’ Henderson and co-workers recently used correlation analysis to define the extent of lattice distortion in images of large patches of purple membrane, and compensated for the distortion by a real space interpolation procedure prior to