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Abstract

We determined the structure of the V₁-ATPase from Manduca sexta to a resolution of 1.8 nm, which for the first time reveals internal features of the enzyme. The V₁-ATPase consists of a headpiece of 13.5 nm in diameter, with six elongated subunits, A₃ and B₃, of approximately equal size, and a stalk of 6 nm in length that connects V₁ with the membrane-bound domain, V₀. At the center of the molecule is a cavity that extends throughout the length of the A₃B₃ hexamer. Inside the cavity the central stalk can be seen connected to only two of the catalytic A subunits. The structure was obtained by a combination of the Random Conical Reconstruction Technique and angular refinements. Additional recently developed techniques that were used include methods for simultaneous translational rotational alignment of the 0° images, contrast transfer function correction for tilt images, and the Two-Step Radon Inversion Algorithm.