Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase.

Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozenhydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na + /K + -ATPase, rabbit skeletal Ca 2+ -ATPase and scallop Ca 2+ -ATPase in order to evaluate the potential for subnanometer resolution as well as the robustness in the presence of disorder. We found that FourierBessel methods perform better for well-ordered samples of skeletal Ca 2+ -ATPase and Na + /K + -ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca 2+ -ATPase, which was too disordered to analyze by Fourier-Bessel methods.