Review: electron crystallography: present excitement, a nod to the past, anticipating the future

From a modest beginning with negatively stained samples of the helical T4 bacteriophage tail, electron crystallography has emerged as a powerful tool in structural biology. High-resolution density maps, interpretable in terms of an atomic structure, can be obtained from specimens prepared as well-ordered, two-dimensional crystals, and the resolution achieved with helical specimens and icosahedral viruses is approaching the same goal. A hybrid approach to determining the molecular structure of complex biological assemblies is generating great interest, in which high-resolution structures that have been determined for individual protein components are fitted into a lower resolution envelope of the large complex. With this as background, how much more can be anticipated for the future? Considerable scope still remains to improve the quality of electron microscope images. Automation of data acquisition and data processing, together with the emergence of computational speeds of 10(12) floating point operations per second or higher, will make it possible to extend high-resolution structure determination into the realm of single-particle microscopy. As a result, computational alignment of single particles, i.e., the formation of "virtual crystals," can begin to replace biochemical crystallization. Since single-particle microscopy may remain limited to "large" structures of 200 to 300 kDa or more, however, smaller proteins will continue to be studied as helical assemblies or as two-dimensional crystals. The further development of electron crystallography is thus likely to turn increasingly to the use of single particles and small regions of ordered assemblies, emphasizing more and more the potential for faster, higher throughput.