Improved crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) by introducing a phosphomimetic mutation in subunit ɛ

The bacterial ATP synthase (F O F 1 ) 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 O F 1 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 α 3 β 3 γ `core', whereas single δ and ɛ 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 ɛ's C‐terminal domain with a phosphomimetic mutation (ɛS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F 1 that diffract to ∼3.15 Å resolution.