Hi Anita, so you tested your crystals inhouse, any idea how they do at the synchrotron ? Still no diffraction ? Since it's a hexamer I would expect the His-tag to be not so important and would rather rescreen with seeding first to see if any other conditions might result in diffracting crystals. Annealing did not help ? Can you slow down the growth of the crystals ? Another option you could try out is limited proteolysis and see if you get a stable fragment, then purify it via SEC and try it with the initial conditions but also rescreening. How big is your monomer ? Do you have a structural homolog / prediction ? Can you make better guesses what you should trim of by design ?
Jürgen On Apr 8, 2011, at 11:43 PM, anita p wrote: Hi Jürgen I tried it by RT capillary as well as under liquid nitrogen, both dont diffract. I ran those on gel and then I confirmed that it is protein. On the SEC it runs as a hexamer.and the DLS shows that it is polydispersed. with regards Anita On Fri, Apr 8, 2011 at 10:03 PM, Jürgen Bosch <jubo...@jhsph.edu<mailto:jubo...@jhsph.edu>> wrote: 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 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<tel:%2B1-410-614-4742> Lab: +1-410-614-4894<tel:%2B1-410-614-4894> Fax: +1-410-955-3655<tel:%2B1-410-955-3655> http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p <crystals...@gmail.com<mailto: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 ...................... 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/
