Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase

H+-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V1 and Vo domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 μm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23 Å. The Fourier transforms of the untilted images revealed unit-cell dimensions of a = 232 Å, b = 132 Å, and γ = 90°, and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22121 symmetry of the crystals and dimer contacts between the V1 domains.