Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.DOI: http://dx.doi.org/10.7554/eLife.10180.001

Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membraneembedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.cryo-EM | evolutionary covariance | V-ATPase | V/A-ATPase | structure V acuolar H + -ATPases (V-ATPases) are large membrane protein complexes responsible for the acidification of intracellular compartments in eukaryotes. These complexes are essential for processes including receptor-mediated endocytosis, coupled transport, and lysosomal degradation (1). V-ATPases localize to the plasma membrane of some specialized cells where they participate in bone resorption by osteoclasts (2) and urine acidification by the α-intercalated cells of the kidney (3). Malfunction or misregulation of these enzymes results in numerous disorders, including osteopetrosis (4) and renal tubular acidosis (5, 6). Expression of V-ATPases on the surfaces of some tumor cells results in increased tumor invasion and metastasis (7). The V/A-ATPase from the thermophilic eubacterium Thermus thermophilus is homologous to the eukaryotic V-ATPase and has a similar overall structure, but is smaller and simpler and functions in vivo as an ATP synthase. V-and V/A-ATPases have similar subunit folds and arrangements of subunits (8-11). The simplified architecture and superior stability of the T. thermophilus V/A-ATPase make it an ideal model to study the structure and function of V-ATPases. Both enzymes are composed of a soluble V 1 catalytic region and a membrane-embedded V O region. The V/A-ATPase subunits I, L, and C in T. thermophilus are homologous to the a, c, and d subunits in eukaryotic V-ATPases, respectively, and, for clarity, the eukaryotic subunit names are used here. With this convention, the T. thermophilus V/A-ATPase contains subunits A 3 B 3 DE 2 FG 2 ac 12 d whereas the eukaryot...

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted F O region is coupled to ATP synthesis in the catalytic F 1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the F O region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c 8 -ring within the F O region support a Brownian ratchet mechanism for protontranslocation-driven rotation in ATP synthases.