Dear Urmi, My first question, did you collect a full data set at a synchrotron and processed this data? I think your diffraction is not as bad as you think it is, and you may get 3-3.5 Å data from it. I asume the crystal structure of the antigen is known and even with 4.5 Å data you could run molecular replacement to define the epitope, which usually is already very important information. You could also try to collect data from a very small crystal. When there is long-range disorder, small crystals may diffract better. Some microfocus beamlines have the possibility to scan the crystal, to find the region in the crystal with best diffraction. Local differences within a crystal can be dramatic.
Some suggestions for crystallization: Add cryoprotectant to your crystallization setup, so you can freeze your crystal directly from the drop where it grew and it does not get any shocks when adding cryoprotectant after it is grown. Also, as you said, test diffraction with unfrozen crystals. Reduce the protein concentration to slow down crystal growth, maybe combined with seeding. Add some trypsin to your crystallization drop to maybe clip off some flexible loops. Good luck! Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Urmi Dhagat Gesendet: Freitag, 24. Mai 2013 04:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Improve diffraction ...any ideas? Hi, I am working on a protein antibody complex which readily crystallizes (crystals form overnight and grow over 2-3 days) in 0.1M Bicine pH 9, 10 % PEG8000. The crystals are chunky - shaped like a parallelogram but they diffract poorly to about 8 Å. I have tried the following to improve diffraction: 1. Screen different temperatures 4°C - crystals have bad form and 10°C crystals grow slower but diffraction does not improve. 2. I have done an additive screen - A few hits came up like Yttrium Chloride and Acetonitrile but they don't improve diffraction either 3. I have tried streak seeding this does not help either 4. Tested different cryo protectants - MPD, PEG400, Ethylene glycol and glycerol - 10 - 15% glycerol seems to work best 5. Not sure if cryo protectant affects diffraction in this case - I will look at room temp diffraction soon to rule this out. 6. Typical diffraction images attached Does anyone have suggestions on what I could try to improve diffraction of my crystals? Urmi Dhagat St Vincent's Institute
