Hi, We are trying to crystallize (hanging drop vapor diffusion method, at 20degC) a 40kDa enzyme (conc. = 20mg/mL) using 0.1-0.2 M HEPES (pH = 8), 20-30% PEG 4K and we obtain needle-like crystals in 2 days. These needle-like crystals diffract up to 4 Angs but decay in the X-ray beam in spite of using cryo-protectant (30% PEG 4K). The protein requires Zn ions for its biological activity. However, it is unstable and degrades within two days and hence EDTA was added for stabilizing the protein (extends its life to 4-5 days).
With Tris buffer (pH 7.5) micro-crystals were obtained after one month. Use of 1-5mM ZnCl2, 1-10mM CdCl2, 20-200mM MgCl2 resulted in precipitation. Please suggest methods of improving the diffraction quality of the needle-like crystals. best regards, -- Pradeep
