Dear Andre, We had a similar case where the crystals were bigger and diffraction was lousy. Our standard dehydration approaches were not very successful. I suggest reading this - Post-crystallization treatments for improving diffraction quality of protein crystals. Heras B<http://www.ncbi.nlm.nih.gov/pubmed?term=Heras%20B%5BAuthor%5D&cauthor=true&cauthor_uid=16131749>, Martin JL<http://www.ncbi.nlm.nih.gov/pubmed?term=Martin%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=16131749>.Acta Crystallogr D Biol Crystallogr.<http://www.ncbi.nlm.nih.gov/pubmed/16131749#> 2005 Sep;61(Pt 9):1173-80. Epub 2005 Aug 16.
But we were able to get a different crystal form (~2A) with high solvent content (75%) by making a bunch of surface mutants. I am not saying that surface mutagenesis is the last resort but something that you might want to consider. See this paper - http://www.jbc.org/content/early/2012/01/31/jbc.M111.327536 Best Regards Harkewal ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andre Godoy [andre_go...@yahoo.com.br] Sent: Tuesday, October 29, 2013 10:18 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystals with large solvent content -dehydratation 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
