Research Article

High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane

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Science  11 May 2018:
Vol. 360, Issue 6389, eaas9699
DOI: 10.1126/science.aas9699

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Protons find a path

Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane).

Science, this issue p. eaas9699, p. eaat4318; see also p. 600

Structured Abstract


The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions. The chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient, generated by the electron transport chain, into the major energy currency of the cell, ATP. The enzyme is a large (about 600,000 Da, in the monomer state) multisubunit complex, with a water soluble complex (F1) that contains three active sites and a membrane complex (Fo) that contains the proton translocation pathway, principally comprised of the a subunit and a ring of 10 c subunits, the c10-ring (10 in yeast, 8 in mammals). F1 has a central rotor that, at one end, is within the core of F1 and, at the other end, is connected to the c10-ring of Fo. During ATP synthesis, the c10-ring rotates, driven by the movement of protons from the cytosol to the mitochondrion, and in turn, the rotor rotates within F1 in steps of 120o. The rotation of the rotor causes conformational changes in the catalytic sites, which provides the energy for the phosphorylation of adenosine diphosphate (ADP), as first proposed in the binding-change hypothesis by Paul Boyer. The peripheral stalk acts as a stator connecting F1 with Fo and prevents the futile rotation of F1 as the rotor spins within it.


Structural studies of the ATP synthase have made steady progress since the structure of the F1 complex was described in pioneering work by John Walker. However, obtaining a high-resolution structure of the intact ATP synthase is challenging because it is inherently dynamic. To overcome this conformational heterogeneity, we locked the yeast mitochondrial rotor in a single conformation by fusing a subunit of the stator with a subunit of the rotor, also called the central stalk. The engineered ATP synthase was expressed in yeast and reconstituted into nanodiscs. This facilitated structure determination by cryo–electron microscopy (cryo-EM) under near native conditions.


Single-particle cryo-EM enabled us to determine the structures of the membrane-embedded monomeric yeast ATP synthase in the presence and absence of the inhibitor oligomycin at 3.8- and 3.6-Å resolution, respectively. The fusion between the rotor and stator caused a twisting of the rotor and a 9° rotation of the c10-ring, in the direction of ATP synthesis, relative to the putative resting state. This twisted conformation likely represents an intermediate state in the ATP synthesis reaction cycle. The structure also shows two proton half-channels formed largely by the a subunit that abut the c10-ring and suggests a mechanism that couples transmembrane proton movement to c10-ring rotation. The cryo-EM density map indicates that oligomycin is bound to at least four sites on the surface of the Fo c10-ring that is exposed to the lipid bilayer; this is supported by binding free-energy molecular dynamics calculations. The sites of oligomycin-resistant mutations in the a subunit suggest that changes in the side-chain configuration of the c subunits at the a-c subunit interface are transmitted through the entire c10-ring.


Our results provide a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase. The structure provides an understanding of the mechanism of inhibition by oligomycin and suggests how extragenic mutations can cause resistance to this inhibitor. The approach presented in this study paves the way for structural characterization of other functional states of the ATP synthase, which is essential for understanding its functions in physiology and disease.

Structure of the monomeric yeast ATP synthase, as determined by cryo-EM, shown as a ribbon diagram.

The subunits are shown in different colors. The F1 complex is located at the top center and is composed of six subunits forming a nearly spherical structure and three subunits comprising the central stalk, or rotor. The Fo complex is located at the bottom, with the identity of the c10-ring clearly seen. The peripheral stalk, or stator, is on the left, and the rotor is in the center of the molecule, extending into F1.


Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo–electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo. Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.

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