 |
 |
Thanks for all the suggestions. Ill try these and post an update here regarding what happens. Thank you so much !! Mahesh On Wed, Jan 22, 2014 at 1:23 PM, Mark van der Woerd <[log in to unmask]>wrote: > 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 <[log in to unmask]> > To: CCP4BB <[log in to unmask]> > 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 >