Hi Catherine, first of all, I'm sorry that you're feeling so frustrated.
In addition to the many sensible suggestions that you're surely going to read here, I would like to point you to a paper I read a while ago in Nature: "In situ proteolysis for protein crystallization and structure determination", http://www.ncbi.nlm.nih.gov/pubmed/17982461 Sometimes proteases can remove from the target protein just those external bits that hinder crystallisation. I would try everybody else's suggestions before attempting this, but here it goes just in case everything else fails. Good luck, Jon On 16 July 2014 16:13, Chris Fage <cdf...@gmail.com> wrote: > Hi Catherine, > > If they are indeed salt crystals, have you tried preparing different > truncations of the gene encoding your target? I've seen a number of > successes in which, after a codon or two was added to the termini of the > gene being overexpressed, the target crystallized beautifully. > > Best, > Chris > > > On Wed, Jul 16, 2014 at 9:55 AM, Bishop, Catherine E. <cati...@ou.edu> > wrote: > >> I have been attempting to obtain a protein crystal of my protein for >> just over 2 years at this point. We have attempted removing the tag, >> binding the protein to its ligand, removing as much salt as possible >> (crashes at at too low a salt concentration)--this lead us to try reverse >> vapor diffusion--seeding, additive trays, optimization around the >> conditions an ortholog of one of the domains crystallized well in, and a >> plethora of other methods. We do get crystals, but they all diffract as >> salt. Most of these salts are found in wells containing a cation (ie. >> lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride, >> etc.). When crystals are too small to shoot, I do set up optimized trays; >> however, if I get larger crystals, they diffract as salt as well. Most of >> these set ups, at this point, have also been conducted in hands other than >> mine and at other facilities known for their successful crystallization of >> proteins. >> >> Has anyone else run into this problem and seen light at the other side?? >> Any suggestions are appreciated. >> >> > -- Dr Jon Agirre York Structural Biology Laboratory / Department of Chemistry University of York, Heslington, YO10 5DD, York, England http://www.york.ac.uk/chemistry/research/ysbl/people/research/jagirre/ +44 (0) 1904 32 8253
