Crystallization and preliminary X-ray analysis of human carbonic anhydrase III

Carbonic anhydrases catalyze the interconversion of carbon dioxide to bicarbonate. Human carbonic anhydrase isozyme III with a C-terminal hexahistidine tag was overexpressed in Eschericha coli, purified and crystallized. Diffraction data (93.4% completeness) were collected to 2.2 Angstrom resolution on an in-house R-AXIS IV++ image-plate system with Osmic mirrors and a Rigaku HU-H3R CU rotating-anode generator operating at 50 kVand 100 mA. A 60degrees sweep of data were collected from a single crystal with a crystal-to-detector distance of 150 mm and a 0.5degrees oscillation angle per frame using an exposure of 60 s per frame at 293 K. The crystals were shown to conform to the Laue hexagonal crystal system P6, with unit-cell parameters a = 44.7, c = 222.5 Angstrom and a scaling R-sym of 0.087 for 11 962 unique reflections. Using the known crystal structure of the rat form of carbonic anhydrase isozyme III, a molecular-replacement model was built. This model was used for rotation and translation searches and uniquely defined the space group as P6(5). Rigid-body refinement of the model was used to generate an initial phased electron-density map with an R-work of 31.17%.