Pradeep, I have similar problem-needles. Seeding/MMS is one method most would suggest. I got good crystals but they appeared fast and they never diffracted (not a single spot while needle diffract 4.5A but hard to pick them as they are very thin) but it might work for you. You could try different cryo, as one time for me 25% glycerol had better spots than 25% ethylene glycol though both were clean cryo. Try adding Zn at expression stage, I don't remember but I have read in literature. Good luck
Date: Sat, 12 May 2012 17:02:39 +0530 From: [email protected] Subject: [ccp4bb] Improvement of diffraction quality To: [email protected] 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
