You might be killing crystals with the cryosolvent. Try soaking the crystals briefly in well solution + 25-30% glucose or sucrose prior to flash cooling. You can also try other cryoprotectants: glycerol, EG, etc. You can also try sequential brief soaks in gradually increasing cryoprotectant concentration. This sometimes works when a direct dunk does not. Or paratone oil.
Roger Rowlett
Hi CCP4BBers,
I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein.
Thanks,
Lucas