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 TmAFP with N-iodosuccinimide. Native and iodinated TmAFP produced two different crystal forms when crystallized using the hanging-drop vapor-diffusion technique. Native crystals were rectangular plates that diffracted to ∼2.5 Å resolution. They were monoclinic and belonged to the space group P2₁, with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 Å, β = 97.0°. Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be collected to ∼1.4 Å. These crystals belonged to the space group P6₁ (or P6₅), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 Å. There were two molecules per asymmetric unit, which corresponds to V_m = 2.46 Å Da⁻¹ and 51% solvent content. A twofold non-crystallographic symmetry was evident from self-rotation calculations.