Crystal structure determination of Escherichia coli ClpP starting from an EM-derived mask

Large ATP-dependent proteolytic complexes carry out the majority of intrace llular proteolysis. To begin to understand the function of these proteases at a structural level, we have combined the information from a number of bi ophysical techniques such as electron microscopy (EM), small-angle scatteri ng, and x-ray crystallography. In this study, we exploited the inherent sym metry of Escherichia coli ClpP, the proteolytic component of the ClpAP/XP A TP-dependent protease, to determine its x-ray crystal structure to 2.3-Angs trom resolution starting with a phase set derived from a low-resolution mas k obtained from EM and small-angle x-ray scattering analysis. Sevenfold and 14-fold noncrystallographic symmetry averaging facilitated phase extension beyond 20 Angstrom and in combination with mask redetermination and matrix refinement was sufficient for completely determining the structure. The st ructure of ClpP is a homo-tetradecamer composed of two heptameric rings enc losing a cavity of similar to 50 Angstrom in diameter that compartmentalize s the 14 serine proteolytic active sites. Comparison of the ClpP structure with those of the 20S proteasome and HslV reveals a striking example of evo lutionary convergence, despite them being unrelated in sequence and fold. M oreover, similarity in their overall architecture suggests a common model f or their action. (C) 1998 Academic Press.