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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Oct 1;68(Pt 10):1229-33. doi: 10.1107/S1744309112036718. Epub 2012 Sep 28.

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

Author information

1
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Abstract

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 ε 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 ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

PMID:
23027754
PMCID:
PMC3490465
DOI:
10.1107/S1744309112036718
[Indexed for MEDLINE]
Free PMC Article

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