Open AccessImproved crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) by introducing a phosphomimetic mutation in subunit ɛ
Author(s) -
Roy Ankoor,
Hutcheon Marcus L.,
Duncan Thomas M.,
Cingolani Gino
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112036718
Subject(s) - escherichia coli , atp synthase , atpase , protein subunit , crystallization , crystallography , catalysis , electrochemical gradient , resolution (logic) , biology , chemistry , biochemistry , stereochemistry , enzyme , membrane , gene , organic chemistry , artificial intelligence , computer science
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.