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

Large ATP-dependent proteolytic complexes carry out the majority of intracellular proteolysis. To begin to understand the function of these proteases at a structural level, we have combined the information from a number of biophysical techniques such as electron microscopy (EM), small-angle scattering, and x-ray crystallography. In this study, we exploited the inherent symmetry of Escherichia coli ClpP, the proteolytic component of the ClpAP/XP ATP-dependent protease, to determine its x-ray crystal structure to 2.3-A resolution starting with a phase set derived from a low-resolution mask obtained from EM and small-angle x-ray scattering analysis. Sevenfold and 14-fold noncrystallographic symmetry averaging facilitated phase extension beyond 20 A and in combination with mask redetermination and matrix refinement was sufficient for completely determining the structure. The structure of ClpP is a homo-tetradecamer composed of two heptameric rings enclosing a cavity of approximately 50 A in diameter that compartmentalizes the 14 serine proteolytic active sites. Comparison of the ClpP structure with those of the 20S proteasome and HslV reveals a striking example of evolutionary convergence, despite them being unrelated in sequence and fold. Moreover, similarity in their overall architecture suggests a common model for their action.