Hi Pradeep, On the stability issue:
Roche sells an EDTA-free protease inhibitor cocktail (other companies probably also do). It is very effective. You can figure out the lowest needed concentration and add it to your protein stock solution. On the other hand, if you can accelerate the nucleation of the crystals (by seeding, or by optimizing the growth condition), you can also minimize the loss caused by proteolysis, as it is unlikely that the contaminant proteases can enter the crystals formed by 40kD proteins. If the instability is not simply caused by proteolysis, then I would suggest to try an extensive seeding experiment. The crystal growth often competes the unwanted aggregation. If you know your enzyme has a substrate, adding the substrate or its inhibitors may greatly help stabilizing it too. On the ion issue: With metal ions such as Zn and Cd, you have to be very careful with the pH and the anions. This was just discussed on the CCP4BB this or last week. pH 8 and 7.5 are almost certain to give you problems with Zn. Can you lower the pH a bit? Ksp Zn(OH)2=3x10^-17(http://www.ktf-split.hr/periodni/en/abc/kpt.html). Note that the hydroxide is multiplied twice in the calculation. So, do not give up too early thinking that the precipitates are your protein instead of Zn(OH)2 and that you have nothing good left in the drops. In many cases crystals even grew in drops with true protein precipitates. Again, try seeding. If you have a rather tight ion binding site (likely true for Zn, as physiological plasma or blood Zn concentration is only about 10 uM), instead of adding the ions to the precipitant solutions, maybe it is better to prepare your protein solution with an equal (or 1-2 fold higher) concentration of Zn or Cd ( 500uM = 20mg/mL of 40KD). The majority of the ions are going to be chelated by the protein's binding sites, leaving little (depending on Kd, OFC) free ions for the hydroxide. The resulting occupancy in the crystal? May not be too low I guess. The other option is to add the Zn or Cd to the crystal only before freezing or shooting, i.e., socking. If you insist using EDTA, then add a little more in your calculation for saturating the EDTA. BTW, I do not think 30% 4K can normally be an effective cryoprotectant. Did you mean glycerol? You might also want to try shooting at RT, so that you know if the poor diffraction is really caused by bad freezing. I often need to dope a little (1-5%) glycerol in the growth conditions to avoid excessively shocking my crystals during cryosoaking. At last but not the least, 4A for a thin needle may not be hell at all, maybe a very hot mirco beam at synchrotron will bless you. Zhijie From: Pradeep Mandal Sent: Saturday, May 12, 2012 7:32 AM To: [email protected] Subject: [ccp4bb] Improvement of diffraction quality 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
