JOURNAL OF STRUCTURAL BIOLOGY 105, 67-74 (1990) Configuration of Subunits within Crystals of Na, K-ATPase Maintained in the Frozen-Hydrated State MANOJ MISRA,~ H. C. BEALL, KENNETH A. TAYLOR, AND H. P. TING-BEALL The Department of Cell Biology, Box 3011, Duke University Medical Center, Durham, North Carolina 27710 Received April 2, 1990, and in revised form August 17, 1990 Two-dimensional crystalline sheets of Na, K-ATPase were studied in the vitrified, frozen-hydrated state by electron microscopy and image processing. The tech- nique of correlation averaging was used to determine the projected structure. The projection map shows asymme- try between the pair of “oLP” protomers comprising a dimer of Na, K-ATPase molecules. The two protomers differ in overall density as well as in shape. One protomer has an oblong shape, whereas the other with higher den- sity has a head and a hook region. Such an asymmetry has not been reported by other laboratories. This asym- metry may either be due to the coexistence of two differ- ent conformations of the enzyme in the dimeric form or due to the simultaneous existence of two molecular spe- cies of Na, K-ATPase. o Iwo Academic PWS. IX. INTRODUCTION The Na, K-ATPase is present in all eukaryotic cells and maintains an electrochemical gradient across the plasma membrane by utilizing the chem- ical energy of ATP hydrolysis to transport Na+ ions out of, and K’ ions into, the cell. The electrochem- ical gradient thus produced provides the potential energy necessary for the execution of various cellu- lar functions among which are the uptake of nutri- ents, water movement, and the maintenance of cel- lular volume. The detailed molecular description of the Na, K- ATPase is necessary for understanding the mecha- nism of ion transport. Several advances toward this understanding have recently been made. The Na, K-ATPase is a dimer composed of two different poly- peptide chains, termed “CY”and “p.” The amino acid sequence of both chains has been determined from complementary DNA (Kawakami et al., 1985, Shull et al., 1985, Ovchinnikov et al., 1986, and Shull et al., 1986, Noguchi, 1986). In addition, two-dimensional crystals of Na, K-ATPase have been formed under several different sets of conditions (Skriver et al., ’ To whom all correspondence should be addressed. 1981; Mohraz and Smith, 1984) thereby opening the possibility that a detailed 3-D structure can be de- termined. Crystals of the Na, K-ATPase can be formed with one or two protomers, each comprising one (Y and one p polypeptide chain, per unit cell. The application of digital image processing to electron micrographs of negatively stained dimer crystals has resulted in 3-D images of the extramembranous portions of the enzyme (Hebert et uZ., 1985b, Mo- hraz et al., 1987 and Ovchinnikov et al., 1985). A conclusion in common with these studies is the structural similarity of the two protomers forming the dimer. In this paper we present a two-dimensional struc- tural study of the unstained crystals of Na, K- ATPase maintained in their native state, in vitreous ice. Our crystals are obtained from phospholipase AZ-treated membranes in the presence of sodium metavanadate. To compensate for the relatively small size and slight disorder within the crystals we chose the technique of correlation averaging to pro- duce an averaged image of Na, K-ATPase crystals in the frozen-hydrated state. No structural symmetry was imposed during the computations of these aver- aged images. Further, since the crystals were main- tained in the frozen-hydrated state, there are no artifacts due to staining; the projected density arises from the entire expanse of the native structure of the dimers forming the crystalline sheet. Our computed two-dimensional averaged images of the ATPase consistently reveal an asymmetry between opposing protomers within each unit cell. This ob- servation confirms that made on negatively stained preparations of these crystals (Beall et al., 1988). The differences between protomers within the dimer may arise from the simultaneous presence of two different conformations or cocrystallization of differ- ent isoenzymes. MATERIALSANDMETHODS Materials Bee venom phospholipase A, was obtained from Sigma; highly purified SDS was from BDH Chemicals (Poole) and fresh pig kid- 67 1047~8477/90 $3.00 Copyright C: 1990 by Academic Press, Inc. All rights of reproduction in any form reserved