Cryoelectron Microscopy of Escherichia coli F1 Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the Complex

Abstract

Monoclonal antibodies directed against epitopes on each of the five subunits (α, β, γ, δ, and ε) of the Escherichia coli F1 ATPase (ECF1 have been prepared and used to localize the subunits in the enzyme complex. Fab' fragments, prepared by pepsin digestion of the antibodies, were bound to ECF1 and visualized by cryoelectron microscopy of the unstained, frozen hydrated ECF1-Fab' complexes. Besides aiding in the identification of the ECF1 subunits, addition of Fabrs to the specimen fortuitously offers additional advantages in this technique. ECF1 labeled with anti-α Fab' is uniformly oriented in the amorphous ice layer, in contrast to unlabeled ECF1 which exhibits a multitude of projection views when examined in ice. Almost all complexes display a triangular projection, which image averaging reveals to be a hexagonal view of ECF1 with Fab' fragments labeling every other peripheral subunit, confirming the alternating arrangement of α and β subunits in the enzyme. A density in the interior of the structure is positioned asymmetrically, adjacent to an unlabeled peripheral mass, indicating that its primary linkage is to a β rather than an α subunit. The composition of the asymmetric density was explored by examining the trypsin-treated ECF1, taking advantage of the unique orientation induced by the binding of anti-α Fab'. Trypsin treatment releases the δ and ε subunits and cleaves the γ subunit; the internal density is reduced but not eliminated, showing the contribution of the γ subunit to the residual structure, and suggesting that the loss of the δ and ε subunits, or a structural rearrangement of the γ subunit, is responsible for its smaller size. Fab' fragments to accessible epitopes on the γ, δ, and ε subunits were localized at the periphery of the hexagonal view of the ECF1, near the unlabeled β subunits. A wide separation of epitopes on the γ and δ subunits (~40 Å) was demonstrated by simultaneous labeling of ECF1 with Fab's directed to these sites. © 1989, American Chemical Society. All rights reserved.