Sample preparation, crystallization and structure solution of HisC fromMycobacterium tuberculosis

Abstract

Histidinolphosphate aminotransferase (HisC; Rv1600) fromMycobacterium tuberculosiswas overexpressed inM. smegmatisand purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space groupP3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using itsCorynebacterium glutamicumcounterpart as a search model. HisC is a dimer in the crystal as well as in solution.