Significance Mechanistic understanding of proteasome function requires elucidation of its three-dimensional structure. Previous investigations have revealed increasingly detailed information on the overall organization of the yeast 26S proteasome. In this study, we further improved the resolution of cryo-EM structures of endogenous proteasomes from Saccharomyces cerevisiae . These structures reveal two distinct conformational states, which appear to correspond to different states of ATP hydrolysis and substrate binding. This information may guide future functional analysis of the proteasome.