This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Proteasomes are large dynamic protein complexes existing inside all eukaryotes and archaea, as well as in some bacteria. The major function of the proteasome is to degrade misfolded or damaged proteins by breaking peptide bonds in an ATP-dependent manner. The polymerized ubiquitin chain acts as a degradation signal that carries the target proteins to the proteasome, where the substrate is proteolytically broken down. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into amino acids and used in synthesizing new proteins. The ubiquitin-proteasome degradation pathway is essential for many cellular processes, including cell-cycle regulation, DNA repair, apoptosis, signal transduction, and protein quality control. We are going to examine the structures of Bovine 26S proteasomes by single-particle cryo- EM. The specific aims for this proposal are: 1) to solve and compare the structures of single- capped 26S proteasome and double-capped 30S proteasome at subnanometer resolution;2) to compare the solution structure of non-capped 20S by cryo-EM with the X-ray structure of 20S;3) to further elucidate the gate opening mechanism of the 26S proteasome by comparing the cryo-EM structures of 26S and 30S with the known structure(s) of 20S, in which the gate in [unreadable] ring(s) remains closed to block the free entry of even unfolded substrates.