2-DIMENSIONAL CRYSTALLIZATION OF CA-ATPASE BY DETERGENT REMOVAL

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca -ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental cond itions. A lipid-to-protein ratio below 0.4 w/w was required for crysta l formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilam ellar tubes were generated upon slow or fast detergent removal, respec tively. Electron crystallographic analysis indicated unit cell paramet ers of a = 159 Angstrom, b = 54 Angstrom, and gamma = 90 degrees for b oth types of crystals, and projection maps at 15-Angstrom resolution w ere consistent with Ca-ATPase molecules alternately facing the two sid es of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both require d the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 Angstrom, b = 77 Angstrom, and gamma = 90 degrees), while maintaining a bipolar orientation of the m olecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a l arge hydrophilic cytoplasmic domain.