Crystallization and preliminary X-ray analysis of the Thermoplasma acidophilum 20S proteasome in complex with protein substrates.

The 20S proteasome is a 700 kDa barrel-shaped proteolytic complex that is traversed by an internal channel which widens into three cavities: two antechambers and one central chamber. Entrance to the complex is restricted by the narrow opening of the channel, which only allows unfolded substrates to reach the active sites located within the central cavity. The X-ray structures of 20S proteasomes from different organisms with and without inhibitors bound have led to a detailed knowledge of their structure and proteolytic function. Nevertheless, the mechanisms that underlie substrate translocation into the 20S proteasome and the role of the antechambers remain elusive. To investigate putative changes within the proteasome that occur during substrate translocation, ;host-guest' complexes between the Thermoplasma acidophilum 20S proteasomes and either cytochrome c (cyt c) or green fluorescent protein (GFP) were produced and crystallized. Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 116, b = 207, c = 310 A (cyt c) and a = 116, b = 206, c = 310 A (GFP), were formed and X-ray diffraction data were collected to 3.4 A (cyt c) and 3.8 A (GFP) resolution.