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

The bacterial ATP synthase (F_OF₁) 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 ∼529 kDa), E. coli F_OF₁ represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F_O) and a peripheral catalytic complex (F₁). The ATPase activity of isolated F₁ is fully expressed by the α₃β₃γ `core', whereas single δ and [epsilon] subunits are required for structural and functional coupling of E. coli F₁ to F_O. In contrast to mitochondrial F₁-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 F₁-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 F₁ that diffract to ∼3.15 Å resolution.