I don't know if there is anyone who has met a problem like this. I have screened a protein and luckily got a hint, I have tried many methods to optimize and to get better diffraction crystals. Every time I have finished with my protein and purified by the same methods and the same buffers, no matter how hard I try, I can not reproduce crystals anymore. I have to screen again, and may find other different conditions. I feel confused, is there anyone who has met this kind of problem? And how can I fix it?
Dear Lian,
there might be many reasons for being unable to reproduce crystals. Did you run your first screen with a robot or by hand? should your answaer be the first, then how big was your drop? I have had many troubles in reproducing xtals grown from drops smaller than 80nL. Therefore the first setup is always 120nl.
In case you are screening by hand, fast equilibration/evaporation should not be your problem.
How old were the solution of your first screen? sometimes the reagents can dissolve ions trapped in the plastic storage tubes, rendering impossible to exactly reproduce the initial conditions.
When you say "I have to screen again" do you always use the same screning reagents? in that case, unreproducibility might sound odd. How much different are your positive hits? can you find any trend you might follow for building an optimization grid?
Anyway, there are few nice books on protein crystallization, and if you search in the archives of Acta Crystallographica D (Biological Macromolecules) you can find many nice papers by (among others) Stura, McPherson, Bergfors.
I hope this helps
Adriana Erica Miele
Go real time and 2D XRD Microscopy! It will help you ID more details of the nano-structure and possibly lead you to the processing "key" in growing consistent quality crystals.
See example of ZnSe single crystal: http://www.youtube.com/watch?v=ykSjd2ZZn64&list=PL7032E2DAF1F3941F
Certainly you have to pay much attention to the crystallization step itself (just like Adriana well tells you), an updated (2013) review is
I. Russo Krauss, A. Merlino, A.Vergara, F. Sica "An Overview of Biological Macromolecule Crystallization" Int. J. Mol. Sci. (2013) 14, 11643-11691
But also to the reproducibility of your protein sample is important, especially if aggregation can be an issue. You will get a single crystal if you have one protein aggregation state in solution. Better you check by DLS or by any other technique providing the size distribution of your sample.
A colleague of mine faced this issue before. He produced crystals only once at 2250uM in an isopropanol condition and failed to reproduce them ever since.
Good news was, he could still solve the structure to3.0A from a single crystal via MR.
From his experience, I would point to the protein purification methods. A slight carry over of a buffer component from a previous step or a cross-contamination might have served as additive in crystallization.
Do you have some crystal matter left from the original hit? You can try to seed with that...
Also keep in mind that commercial screening kit conditions can be hard to reproduce. Because your stock solutions might be less consistent with the stocks used in the screen. Broaden up your follow up screen, maybe the shiny nice single rock is just around the corner... :D
good luck!
S.
Thanks for all your comments ,Adriana Miele，Ravi Ananth，Alessandro Vergara and Stefan Gajewski . It's a very late reply but your advice is very useful. I have solved the reproducable problem through mutation, and now the crystals could be reproduced very well, but now I meet the new problem, my crystals can not be diffracting well.
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