Hi Morgan, Some of the things that you could try with the conditions that you have already found:
1) Lower the temperature for incubation, which might slow down crystal formation and decrease potential defects as the crystals form 2) Harvest the crystals using a humidity control device like the Watershed from MiTeGen 3) Try adding oil to the top of the well and harvest through the oil as cryoprotectant 4) Try different cryos and different methods of adding cryoprotectant 5) Try collecting at room temperature – that may help identify if the issue is the crystals or the cryo Finally, you may need to find other conditions in which your complex forms crystals. Feel free to reach out to the National Crystallization Center at HWI if you need assistance with finding new conditions (www.getacrystal.org). Good luck! Sarah Sarah EJ Bowman PhD Associate Investigator | Hauptman-Woodward Medical Research Institute Director | National High-Throughput Crystallization Center p: +1 716 898 8623 e: sbowman at hwi.buffalo.edu Research Webpage<https://hwi.buffalo.edu/scientist-directory/sbowman/> Crystallization Center Webpage<http://www.getacrystal.org> Hauptman-Woodward Medical Research Institute 700 Ellicott Street | Buffalo, NY 14203 From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of "Blake, Morgan Elizabeth" <more...@uab.edu> Reply-To: "Blake, Morgan Elizabeth" <more...@uab.edu> Date: Wednesday, November 22, 2023 at 11:54 AM To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Fragile Crystals Hello! I am a PhD student working on a crystallography project to wrap up my dissertation research. I have purified a complex of two proteins, and I can consistently grow crystals in 10% PEG3350, Warning! This message was sent from outside your organization and we were unable to verify the sender. sophospsmartbannerend Hello! I am a PhD student working on a crystallography project to wrap up my dissertation research. I have purified a complex of two proteins, and I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow to a large size (greater than 0.5 mm), but the crystals seem to be very fragile. When we open the drops to harvest the crystals, we have little time to harvest the crystals before they crack. When we move the crystals to a cryoprotectant, over time they start fracturing. We've tried using different percentages of glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, the crystals do not diffract well, with spot patterns that look very streaky/mosaic, which I presume is due to the defects that we see in harvesting/handling. We have screened for alternate crystallization conditions, but we seem to get the same morphology in other conditions. Does anyone have suggestions for additives we could use post-crystallization to help stabilize our crystals? Thanks for your advice! ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
