X-Ray Crystallography of Sugar Related Proteins

Abstract

Saccharides are essential for inter- and intracellular functions. In order to understand the molecular mechanisms of the synthesis and recognition of saccharides, it is important to obtain three-dimensional structures of protein-saccharide complexes. X-ray crystallography is the most efficient and widely used method for determining the structures of proteins and their complexes. Because a large quantity of protein sample must be prepared for crystallization, recombinant DNA techniques are used to overexpress target genes in bacterial, insect, or mammal cell cultures. From several liters of cultured cells, several milligrams of protein sample can be purified by a series of chromatographic steps. Subsequently, crystallization conditions are screened, and the resultant crystals are irradiated with X-ray beams. Ultimately, the structure is determined based on the X-ray diffraction pattern. Phase information, which is essential for structure calculation, can be obtained from previously determined structures of similar proteins, heavy metal-bound crystals, or crystals of the selenomethionyl protein. In addition, a new technique for determining phase using selenium-containing saccharides is described.