I'd say since you obtained crystals with your tag it is not a disturbing factor and either disordered or making contacts. So removing the tag you might end up not getting crystals in the worst case. Now to the question why they don't diffract. Did you test the old fashioned way at RT in capillary ? Maybe your freezing is the problem. The inability to purify TEVed protein suggests that you have at least a dimer perhaps or higher multimer. Since you observe three bands on the gel, uncleaved, cleaved and TEV itself. Any idea about DLS or migration behavior on a SEC column for your uncleaved protein ? Since you have crystals what I would do is crush them up and rescreen whatever commercial screens you have available using some of the crushed crystals as seed. A second option would be to take your current condition and modify it with say the most frequent 12 cryo solutions added in maybe 5-10% effective concentration and see if you still obtain crystals. Before freezing them I would then freeze directly from the drop and a second crystal would be soaked into whatever concentration of the cryo is required to properly freeze. I bet you will find something that works. Of course you don't stop there. Once your crystals are on the gonio and diffract or don't diffract you will flash anneal them once or multiple times and report back in a nice table which condition resulted in the 2.3A dataset. So you have roughly 146 experiments to do alone from cryo- optimization. Good luck ! Jürgen
...................... Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p <crystals...@gmail.com> wrote: > Hi Crystallographers, > I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage > site. > I am getting crystals with the his tag and tev site intact, but they dont > diffract. > Is it probable that they dont diffract because of the extra his tag and the > tev site? > > I am trying to get rid of this tag but the reaction is optimum at 10:1 > protein to TEV ratio in micrograms overnight incubation without shaking. > I tried to run it on histrap column after this reaction but I am not able to > purify cleaved protein from TEV and uncleaved. > I have tried several times but I get 3 bands ie., the TEV, uncleaved and > Cleaved. > I have also tried to use the Nibeads instead of the histrap column, but no > difference is seen. > Is there a possible way to approach this problem? > > Suggestions awaited > Anita
