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Two-dimensional membrane crystals of renal Na,K-ATPase were analyzed by electron microscopy and image processing. The particular property of the crystals in this work was that they showed unit cell parameters similar to the previously studied p21 crystals but lacked the dyad axis as observed in nominal O"-projections. A three-dimensional reconstruction revealed that structural differences between ag-units of the enzyme gave rise to the asymmetry. A high degree of two-fold rotational symmetry was observed in the middle of the structure while the protein units had different three-dimensional shapes at levels above and below the central sections. The simultaneous coexistence of different forms of Na,K-ATPase suggests that the conformational flexibility of the enzyme plays an important role in the pumping process.

Two-dimensional membrane crystals were induced in purified preparations of membrane-bound Na,K-ATPase. The crystals were analyzed by cryo-electron microscopy in frozen-hydrated preparations using minimal dose conditions and elastic brightfield. The vanadate-induced crystals showed predominantly p1 or p21 symmetries and image analysis using correlation averaging demonstrated that the protomers in dimeric crystals were either similar or different in the projected structure. The observations suggest that the Na, K-ATPase protomes are gradually reorganized within the membrane crystals during the assembly process.