ABSTRACT
For an understanding of the function of many biological particles, knowledge of their threedimensional (3-D) architecture is a prerequisite. For many smaller molecules, the answer can often be found using the techniques of X-ray crystallography, which are capable of resolving structure
to the atomic level.1•2 Electron microscopy of noncrystalline biological specimens, however, yields structural information at a lower resolution, typically in the range of 2 to 3 nm in three dimensions; and although images of two-dimensional crystals have been recorded in the electron microscope with a resolution of 0.3 nm,3·4 such fine details cannot yet be resolved in images of single particles. The advantage of electron microscopic structure determination of single particles lies in the fact that no crystallization is necessary and virtually all larger macromolecules are amenable to such an approach. In a preparation of single molecules, no binding sites are blocked (e.g., by a crystalline arrangement), which make this approach uniquely suitable for studying biological function. One of the best examples in the reconstruction of the Escherchia coli ribosome, where first the two subunits have been reconstructed followed by the complete ribosome. Constructs of the ribosome with tRNAs bound have been analyzed and these studies continue so that all ligands will be localized in the ribosomes at different stages of translation. 5•6
Throughout the development of 3-D reconstruction in electron microscopy, however, one goal has been to determine the architecture of biological particles with higher and higher resolution. There are two major effects that limit resolution. One limit is set by the artifacts that occur during preparation of macromolecules for electron microscopy. The effects of negative staining and subsequent drying have been widely discussed in the literature.7 Only with the use offrozen hydrated preparation techniques8-11 can these artifacts be avoided. A second serious limitation of the resolution is the radiation sensitivity of biological objects.12 Single atoms within some inorganic specimens can be imaged with the electron microscope, but the electron dose required for these images would reduce the biological material to ashes. For biological specimens, extremely low electron doses must be used. In fact, the higher the intended resolution, the lower the electron dose must be in order to avoid destruction of fine structures. It is widely accepted that at resolutions on the order of 2 nm, electron doses of 10 e/A2 are acceptable. At this level, however, the signal-to-noise ratio of a single image is less than 1 meaning that the noise in an image is stronger than the signal.
