Crystallization of mitochondrial creatine kinase on negatively charged lipid layers

Polymorphic forms of crystals of mitochondrial creatine kinase (Mi-CK) octamers were generated by the lipid-layer technique, using cardiolipin as interphase adhesion matrix. Depending on the protein and lipid concentration, different types of monolayers and 3-D stacks thereof assembled in a low ionic strength crystallization buffer. Sodium tungstate was found to promote and stabilize the crystal formation, though in-plane crystallization was also possible in the absence of tungstate. All crystal forms exhibited a p4 symmetry with lattice parameters (a = b) ranging from 10.6 to 24.0 nm and with one or four octamers per unit cell in end-on orientation. In ice-embedded crystals, which showed a molecular packing different from that of negatively stained preparations, structural features of the Mi-CK octamer were observed at a resolution of 1 nm. The crystallization process took advantage of the electrostatic interaction between negatively charged lipid head groups of cardiolipin and positive charges located at the top/bottom faces of the Mi-CK octamer. In the absence of a cardiolipin support, Mi-CK formed linear filaments from a solution of phosphotungstate by association of octamers via their top/bottom faces. When tungstate was used instead of phosphotungstate, the filaments aligned themselves into large crystalline assemblies.