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 Å 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 kV and 100 mA. A 60° sweep of data were collected from a single crystal with a crystal-to-detector distance of 150 mm and a 0.5° 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 Å 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 P6₅. Rigid-body refinement of the model was used to generate an initial phased electron-density map with an R_work of 31.17%.