Optimising Non-diffracting Crystals

In the event your crystals diffract poorly the following tips and tricks to try may be helpful in obtaining better diffracting crystals. It is also important to note that depending on how much protein you have available to you may change which procedures you will try.

  1. If you have crystals from an additive screen or seeding – test their diffraction. If you have access to in-plate screening, you can test the crystals without disturbing them, which will give the best idea of their native diffraction. Perhaps one of the ugly crystals diffracts well enough?


  1. Try cross seeding – seed one or more initial screen(s) (rather than an optimisation). Try initial screening with seeding at different temperatures. If you are currently using vapour diffusion, try microbatch. Or vice versa.


  1. Try in-situ proteolysis. Add a very small amount of protease to your protein sample (chymotrypsin is traditional) – say 1:10,000 or 1:1000 compared to your protein concentration then set up that mixture in initial screens.


  1. Is there a ligand/inhibitor/other small molecule that binds? Add that to your protein before crystallisation. Maybe even try this first!


  1. Lysine methylation/cysteine modification/other side chain modifications.


  1. Try using DSF or some other technique to look at your protein’s stability in the formulation it is in. Maybe you can make happier protein by changing the pH, buffer or salt.


  1. If you want to be a little more rigorous, take your protein, and a number of different proteases, and do a time-course experiment with each protease (add 1:1000 protease to your protein, then take samples at time points – say 0.5 hours, 1 hour, 5 hours and overnight) then run out on a gel (or analyse by MS) and see if you come down to a stable fragment. If you do – then use that protease, and while you are waiting for the crystallisation trials to do their thing, find out what the end points of the proteolysis fragment are, and make that construct.


  1. Try a different expression system (different tag, different position of the tag, cleave/don’t cleave the tag). If the protein is produced in a eukaryotic system (and is glycosylated) try a different one to get a different glycosylation pattern. Try kifunensine treated cells if you are in a mammalian expression system.


  1. Try the same protein from other species