Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor

Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC. An iodinated derivative was prepared by incubating the 8.5 kDa T mAFP with N-iodosuccinimide. Native and iodinated TmAFP produced two differ ent crystal Terms when crystallized using the hanging-drop vapor-diffusion technique. Native crystals were rectangular plates that diffracted to simil ar to 2.5 Angstrom resolution. They were monoclinic and belonged to the spa ce group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 Angs trom, beta = 97.0 degrees. Crystals of iodinated TmAFP formed elongated hex agons that allowed data to be. collected to similar to 1.4 Angstrom. These crystals belonged Co the space group P6(1) (or P6(5)), with unit-cell dimen sions a = 73.85, b = 73.85, c = 53.15 Angstrom. There were two molecules pe t asymmetric unit, which corresponds to V-m = 2.46 Angstrom Da(-1) and 51% solvent content. A twofold non-crystallographic symmetry was evident from s elf-rotation calculations.