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The formation of two-dimensional crystals of the membrane-bound enzyme microsomal glutathione transferase is sensitive to fractional changes in the lipid-to-protein ratio. Variation of this parameter results in crystal polymorphism. The projection structure of a p6 crystal form of the enzyme has been determined by the use of electron crystallography. The unit cell at 3 A resolution is comprised of two trimers. The hexagonal p6 and the orthorhombic p21212 crystal types have common elements in the packing arrangement which imply dominant crystal contacts. An overall structural similarity between the protein molecules in the two crystal forms is suggested by the projection maps. Furthermore, a comparison of the p6 and p21212 projection maps identifies additional corresponding protein densities which could not be assigned to the microsomal glutathione transferase trimer previously. Surprisingly, an ambiguity of the rotational orientation was found for trimers interspersed at certain positions within the crystal lattice., (Copyright 1999 Academic Press.)

Various crystallization parameters were investigated to obtain two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase for structural analysis by electron crystallography. The protein was crystallized by reconstitution of the solubilized trimer into proteoliposomes. Crystallization occurs when minimal amounts of lipid in the range of three lipid molecules per protein trimer are added to the dialysate. Once crystals were obtained, the effect of several parameters on the crystallization was determined. The temperature and initial detergent concentration were found to be crucial parameters in influencing the size of the crystals, and conclusions could be drawn about the rate dependence of the crystallization process. Two highly ordered crystal forms, which are suitable for structural analysis by electron crystallography, were obtained under the two-dimensional crystallization conditions described here., (Copyright 1998 Academic Press.)

Through the use of electron crystallography, it has been possible to obtain high resolution structural information regarding a mammalian protein that spans the lipid bilayer. Two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase were induced by slow detergent removal from a mixture containing low amounts of phospholipid. Images of specimens stabilized in tannin were collected using electron cryomicroscopy. The projection structure at 4 A shows tightly packed trimers of the protein. Each of them contains an inner core of six parallel alpha-helices delineating a central low density region. The helical bundle is partly surrounded by elongated domains.