Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε

The bacterial ATP synthase (FOF1) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of similar to 529 kDa), E. coli FOF1 represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F-O) and a peripheral catalytic complex (F-1). The ATPase activity of isolated F-1 is fully expressed by the alpha(3)beta(3)gamma 'core', whereas single delta and epsilon subunits are required for structural and functional coupling of E. coli F-1 to F-O. In contrast to mitochondrial F-1-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F-1-ATPase catalytic core. Destabilizing the compact conformation of epsilon's C-terminal domain with a phosphomimetic mutation (epsilon S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F-1 that diffract to similar to 3.15 angstrom resolution.