Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.