The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. The 2.5 MDa complex is built of 34 different subunits and comprises two subcomplexes: the 20S core where proteolysis takes place and one or two regulating particles which prepare substrates for degradation. Whereas the structure of its 20S core particle has been determined by X-ray crystallography two decades ago, the structure of the 19S regulatory particle, which recruits substrates, unfolds them, and translocates them to the core particle for degradation, has remained elusive. Recently, the molecular architecture of the 26S holocomplex was determined by an integrative approach based on data from single particle cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. Based on a 6Å structure of the Saccharomyces cerevisiae proteasome, and molecular dynamics-based flexible fitting we were able to generate a near-atomic model of the 26 holocomplex. Furthermore, by applying deep image classification to a very large data set, we defined its conformational landscape.

Electron cryotomography allows to perform structural studies of macromolecular and supramolecular structures in situ, i.e. in their functional cellular environments. We applied electron cryotomography with a new type of a phase plate, the Volta phase plate, to hippocampal neurons grown on EM grids and were able to locate 26S proteasomes with high fidelity and nanometer precision in these cells. Subtomogram, classification and averaging revealed the existence of different states of assembly and conformational states. From this we can infer the activity status of individual 26S complexes.