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Hi Andre, Are you sure that the cryoprotectant has been optimized?..I am sure you must have already done it but just in case..sometimes the cryo protectant which makes the ice rings disappear is not the best one and playing around that concentration as well as different alternatives can have a huge impact. Another thing, again trivial, is to shoot different parts of the crystal. For big crystals sometimes the regions near the tips diffract better than regions in the interior ( plausibly correlated with penetration of the cryoprotectant during treatment as well as other deformities in the central portions that might have occurred during crystal growth or vitrification.) My humble two cents, Best, Arka Chakraborty On Tue, Oct 29, 2013 at 5:17 PM, Danilo Belviso <[log in to unmask]>wrote: > Dear Andre, > > you could try with the protocol described in the following paper > > Acta Crystallogr D Biol Crystallogr. 2013 69,920-3. > Using high-throughput in situ plate screening to evaluate the effect of > dehydration on protein crystals. > Douangamath A, Aller P, Lukacik P, Sanchez-Weatherby J, Moraes I, > Brandao-Neto J. > > It gives very good results with membrane proteins where the water content > is high. > > Danilo > > > On Tue, 29 Oct 2013 08:18:21 -0700, Andre Godoy <[log in to unmask]> > wrote: > >> Dear all >> >> I'm trying to solve a beautiful large crystal that, unfortunately, >> doesn't go further than 5 A resolution. I believe that in this case, >> the lack of resolution is due the high solvent content (about 66%). >> Therefore, my next strategy should be the dehydratation. Yet, I never >> (sucessfully) did that. I read different approachs, were people >> equilibrate crystals in dehydratation solution for days, or do more >> than 20 steps, or add solvents. Since i never had sucess in my trials, >> I was thinking that someone can suggest a protocol (should I remove >> all salt?, should I keep the additive concentration?, how much >> precipitant should I add? how many steps?). >> >> crystal condition: 23% PEG 3350, 0.2M NaCl, 0.1M Tris pH 8.5, 3% >> galactose (orthorhombic crystals, with about 0.6 x 0.6 mm) >> >> all the best, >> >> Andre Godoy >> > -- *Arka Chakraborty* *ibmb (Institut de Biologia Molecular de Barcelona)** **BARCELONA, SPAIN** *