Mahesh, Have you tried to see what happens when you do not freeze the crystals at all? Do you get good resolution data? Do the crystals suffer radiation damage?
Those are two important questions. First, if your crystals do not diffract well before you freeze them, usually (but not always) they will also not diffract well after you freeze them and you can try cryo-conditions until you see blue in the face, but they will never improve. In that case, you need new/better crystals. Second, many well-diffracting crystals do not necessarily need to be frozen at the home source. Of course they do need to be frozen at the synchrotron, but if you can get good (enough) data at home... On to recipes: try increasing dioxane. Try adding alcohols (somewhat similar to dioxane, maybe iso-propanol). Expect this to be a pain: very volatile agents make it difficult to harvest crystals. Of course try the mixtures without protein or crystals for freezing conditions and how well you can mix the components. Salt and organics are often not compatible. Try to regrow your crystals in sodium malonate (replace Ammonium sulfate with malonate). Try to regrow them in the presence of small amounts of the cryo-protectants you have already tried. For the ones that work, repeat with higher amounts. Very often crystals are much happier when you don't soak/tinker with them and freeze them straight out of the drop. Good luck. Mark -----Original Message----- From: Mahesh Lingaraju <[email protected]> To: CCP4BB <[email protected]> Sent: Wed, Jan 22, 2014 10:38 am Subject: [ccp4bb] cryoprotection ideas for salt based condition Hello folks, I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10% dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have finding the right cryoprotection for salt based conditions; I have tried glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing ammonium sulphate concentration in presence of 5-10% glycerol. The crystals disintegrate in any kind of PEG based cryo. I made all these solutions in the mother liquor and tested if they freeze clearly before using them. However when I loop the crystals and try to soak them in these cryo- mother liquor, a lot of salt crystals suddenly form around the protein crystal and I see diffraction only from these salt crystals. The best I have been able to get so far is ~ 9Å diffraction (Home-source) with DMSO as the cryoprotectant. I have also tried using 1 M sodium malonate as the cryoprotectant but my crystals are not too stable in this mother liquor probably because I had to lower the ammonium sulphate by ~ 7-10% to make the drop not form salt crystals instantly when exposed to air. Other than trying to make the crystals more cryo-ready by finding other hits or may be growing the same crystals with some cryoprotectant, I was wondering if any of you have ideas based on your experience or suggestions in case I am doing everything wrong in the first place. Thanks for all the help, Mahesh
