Direct visualisation of conformational changes in EF0F1 by electron microscopy

The isolated H+-ATPase from Escherichia coli (EF0F1) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing. The resulting three-dimensional maps showed that the F-1-part is connected by a prominent stalk to a more peripheral part of F-0. The F-1-part showed stain-accessible cavities inside. In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator. Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-beta,gamma-imidodiphosphate (AMP-PNP) were calculated. Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F-0-part of EF0F1, whereas a major conformational change in the F-1-part was observed. (1) The diameter of the F-1-part decreased upon binding of AMP-PNP mainly in the upper half of F-1. (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F-1-part which was missing in its absence. (3) The stain-accessible cavity inside the F-1-part altered its pattern significantly. (C) 2000 Academic Press.