The ubiquitin-proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Semi. Here, we localize the C-terminal helix of Semi binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with semi deletion. The approximate position of the N-terminal region of Semi bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Semi can assume different conformations in different contexts, which supports the idea that Semi functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer. (C) 2013 Elsevier Inc. All rights reserved.