I am working on Ankyrin repeat domain of a protein and it produces a good shaped crystals. However, at the beginning it was twinned. Thus, I tried to crystallized in low temperature with some additives. Now, twining problem is solved but the diffraction is still low .
Is there any way to get stronger diffraction.
To exactly tell what need to be fixed may not be easy although you may try some of these..
1. Try diffraction at room temp to look for the difference. sometimes poor cryoprotectants may be the reason.
2. Try different cryoprotectants if possible.
3. Try seeding, both micro and macro.
4. Use additives.
6. Try both hanging and sitting drop optimization.
7. Check with DLS for homogeneity of your protein
8. Try changing construct boundaries or cleaving any tag. After this you serach for new conditions.
9. Try annealing and/or dehydration.
10. Try cross-liking.
Do you get to shoot your own crystal? Sometimes taking care to find the best spot to aim for makes a world of difference. Syed has covered most of the bases - beyond this the best advice would be to do a matrix around your current condition and look for less nucleation / more growth of fewer crystals.
For my EpsG crystal we had to lower the precipitant and protein concentration while growing at 5C instead of room temperature - this resulted in crystals taking almost two weeks to grow! Also, we were doing hanging drop so adding some paraffin oil to cover the reservoir slowed it down even further.
Crystallization in the presence of proteases (trypsin, chymotrypsin, papain, subtilisin, elastase or other) may cut off unstructured loops that limit high order packing. This would increase resolution.
Ensure that your protein > 95% pure and then optimize the crystallization condition by adjusting parameters such as pH, concentration of precipitant, protein concentration, and etc.
I presume you mean resolution is low when you state the diffraction is low.
How many amino acids are there in your protein? The bigger the asymmetric unit the harder to get get good resolution. Although, I'm not sure how they were able to crystalize a virus. Check Homologous structures (via Clustaw), if there is a possibility that there may be loops or long helices that connect different domains then good tight crystal packing is hard to achieve which in turn can effect resolution. One trick is to do mutations of an amino acid that will that will " stiffen" a flexible long helix.
Great suggestions listed. In addition to these, other issues - are you using a home source, or a synchrotron? What's the CCD? Are you soaking or co-crystallizing any ligands into the crystal? In regards to cyroprotectants, are you doing a gradual increase in % of, for instance, glycerol, or PEG 400? Keep in mind crystals can be deceiving, they may look great, however properties like mosaicity may be something out of your control.
The above experts have covered almost all the aspects that you can try for improvement of your crystal diffraction. I personally suggest that you might try reductive methylation for the lysines on the surface of your protein and then do crystal screening again. The change of protein surface properties and reduction of protein surface entropy could help with crystallizaton. Some proteins might get tremendous improvement. Good luck!
Syed and other reseachers have covered all the points which helps in improving x-tal diffraction. You can also try mother liqour as cryoprotectant and snap annealing.
Best wishes..
Have you tried collecting data at room temperature to assess if it is your cryo-protectant that is causing the poor diffraction?
Another thing you could try, not so much a way of improving the diffraction from the crystal but improving the data quality, is combining datasets. This may give you a bit of a resolution boost.
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