Cryoelectron microscopy of Escherichia coli F1 adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex.
Monoclonal antibodies directed against epitopes on each of the five subunits (alpha, beta, gamma, delta, and epsilon) 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 Fab's to the specimen fortuitously offers additional advantages in this technique. ECF1 labeled with anti-alpha 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 alpha and beta 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 beta rather than an alpha 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-alpha Fab'. Trypsin treatment releases the delta and epsilon subunits and cleaves the gamma subunit; the internal density is reduced but not eliminated, showing the contribution of the gamma subunit to the residual structure, and suggesting that the loss of the delta and epsilon subunits, or a structural rearrangement of the gamma subunit, is responsible for its smaller size.(ABSTRACT TRUNCATED AT 250 WORDS)
Gogol
E P
EP
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Aggeler
R
R
Sagermann
M
M
Capaldi
R A
RA
D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D003461:Crystallography / Q000379:methods; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D005615:Freezing; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008854:Microscopy, Electron / Q000379:methods; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000302:isolation & purification*; D013194:Staining and Labeling; D013329:Structure-Activity Relationship; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D014357:Trypsin; D006180:Proton-Translocating ATPases