Janet Newman wrote a review on improving diffraction a few years back: http://dx.doi.org/10.1107/S0907444905032130 it is open access.
Probably the most underappreciated aspect of diffraction is the purity of the protien. This is because "impurities" like slightly misfolded versions of your "native" structure can genreally still find their way into the lattice. These are "defects", and the distorition they create will push thousands of other unit cells out of place. So, 95%, or even 99% purity can be "not enough". Getting the molecule to the point where it is the brightest band on the gel is generally "enough" to screen for crystallization conditions, but it is not uncommon for crystals from un-clean protein to diffract poorly. My advice: try adding a column to your purification protocol. Or, better yet, try fractional recrystallization. This is where you use your crystallization condition to "crash out" your entire stock, spin down the crystals, redissolve them, and then maybe do this a few times in a row. Yes, you loose a lot of material, but the stuff you lost is stuff that doesn't crystallize anyway. -James Holton MAD Scientist On Tue, Oct 26, 2010 at 2:31 PM, Matthew Bratkowski <mab...@cornell.edu>wrote: > > Hi. > > Here is some additional information. > > 1. The purification method that I used included Ni, tag cleavage, and SEC > as a final step. I have tried samples from three different purification > batches that range in purity, and even the batch with the worst purity seems > to produce crystals. > > 2. The protein is a proteolyzed fragment since the full length version did > not crystallize. Mutagenesis and methylation, however, may be techniques to > consider since the protein contains quite a few lysines. > > 3. There are not any detergents in the buffer, so these are not detergent > crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. > > 4. Some experiments that I have done thus far seem to suggest that the > crystals are protein. Izit dye soaks well into the crystals, and the few > crystals that I shot previously did not produce any diffraction pattern > whatsoever. However, I have had difficulty seeming them on a gel and they > are a bit tough to break. > > 5. I tried seeding previously as follows: I broke some crystals, made a > seed stock, dipped in a hair, and did serial streak seeding. After seeding, > I usually saw small disks or clusters along the path of the hair but nothing > larger or better looking. > > I also had one more question. Has anyone had an instance where changing > the precipitation condition or including an additive improved diffraction > but did not drastically change the shape of the protein? If so, I may just > try further optimization with the current conditions and shoot some more > crystals. > > Thanks for all the helpful advice thus far, > Matt > > >
