2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy

Abstract

Large, well-ordered 2-D crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H+-ATPase grow rapidly on the surface of a polyethylene glycol-containing mixture similar to that originally developed for growing 3-D crystals of this integral membrane transport protein. Negative stain electron microscopy of the crystals shows that many are single layers. Cryoelectron microscopy of unstained specimens indicates that the crystals have a p6 layer group with unit cell dimensions of a = b = 167 Å. Image processing of selected electron micrographs has yielded a projection map at 10.3 Å resolution. The repeating unit of the ATPase crystals comprises six 100 kDa ATPase monomers arranged in a symmetrical ring. The individual monomers in projection are shaped like a boot. These results provide the first indications of the molecular structure of the H+-ATPase molecule. They also establish the feasibility of precipitant-induced surface growth as a rapid, simple alternative to conventional methods for obtaining 2-D crystals of the integral membrane proteins useful for structure analysis.