Dear all, A very happy new year to all.
I would appreciate some expert advice on optimizing a crystallization condition in which the initial hits were obtained with ethylene glycol as the main precipitant. Here is the summary of things tried. We have a protein, size (31Kda) and the starting protein buffer is 0.1M Tris pH7.5, 0.1M NaCl, 10% glycerol. The initial crystal hit was obtained from the emerald cryo kit condition that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals were tiny (10-20um). A crystallization matrix to obtain better crystals by varying the imidazole pH and ethylene glycol concentrations was tried from which the best condition obtained was 0.1M imidazole pH 6.5 , 30% (v/v) ethylene glycol. The crystals were slightly bigger 50um. On trying the additive screen, bigger crystals (200um) were obtained, but putting them under the x-ray beam with direct freezing did not yield any diffraction spots. Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture also yielded similar results. Low resolution spots near the beam stop were also not seen. Similarly spots indicative of salt was also not seen. It just had hazy ice rings kind of stuff. (The beam was definitely on the crystal) To check if what we have was salt, a control condition with no protein was tried. Also the crystals were run on a gel after thorough washing. Both these tests, show that they are definitely protein crystals and not salt. Seeding also did not yield any improved crystals. I was suggested using di-ethylene glycol, propane diol as alternatives. I would greatly appreciate if you can give your opinion on using other di-alcohols as precipitants or other ways to improve these crystals. I tried searching the PDB to see if someone had actually used ethylene glycol as a precipitant, most of them were used as a cryo condition than actually as a precipitant. Thank you very much in advance. Regards Shankar Srinivasan