I have been growing quite large protein crystals after several rounds of standardization. However good the crystals may look, I am getting very faint diffraction spots from them, that too, around 8-10 angstrom resolution. How may I improve the quality of my protein crystals?
P.S. I have checked that they are not salt crystals
I'd try a combination of the following:
1) Try tweaking your crystallization conditions. There are additive kits that are great for this
2) Try seeding using your initial crystals
3) Try dehydrating your crystals--there are numerous methods that have been described in the literature
4) Try different cryoprotectants and concentrations
Unfortunately there isn't a magic bullet that will definitely work. Good luck!
I did try a few with home-made additives but they didn't work in augmenting diffraction quality of the crystals. I would definitely try working with cryoprotectants next time. Thanks a lot!!
In addition to the above suggestions, here are some further possibilities:
- If you are dealing with a recombinant protein, try different constructs, e.g. by trimming ancillary domains.
- Make sure that you remove any affinity tag
- If known, try to co-crystallize the protein with a ligand
The suggestions from James and Pierre are very good suggestions. One other approach that you might try (perhaps in combination with the suggestion to try different constructs/remove affinity tags) is seeding into a new screen. This has been shown to significantly increase the number of hits obtained, and can access a new, better crystallisation condition.
See Article Random Microseeding: A Theoretical and Practical Exploration...
It is not unusual that nice looking crystals show poor diffraction patterns. From my experience, three of the reason for such behavior are:
- You may use additive kits (=brute force), but if you know your protein, it is better to try specific ligands. For example, human liver FBPase crystallizes very well in presence of AMP because AMP binds the AMP-site and induces a conformational change that favors a crystal form that diffracts well.
- Search for similar proteins in PDB and reformulate your crystallization conditions. It may be of help to try different predictions (check http://ffas.burnham.org/XtalPred-cgi/xtal.pl). In particular I would try to model the protein (check https://www.proteinmodelportal.org) to locate flexible regions and identify similar proteins that have been crystallized.
- If the above fails, the protein becomes a variable in the crystallization screen (doi: 10.1016/S1047-8477(03)00041-8). Think of modifying your protein by site directed mutagenesis, truncation... A “rational approach” relies on searching for differences with homologous proteins that have been crystallized (doi: 10.1038/349541a0), entropy surface reduction (check http://services.mbi.ucla.edu/SER/?job=106140&key=488963) and a combination of luck and patience.
Good luck
In addition to the methods already mentioned here, you can do the crystallization in a weightless (zero-gravity) conditions, such as in an orbiting earth satellite in space, or in a simulated zero-gravity condition on earth (such as those used in training would-be astronauts). Zero-gravity conditions make for well-ordered crystals, which diffract very well (sometimes with less than 1 Angstrom resolution). Vielen Gluck.
As Vicente suggested, zero-gravity may be an option, but from my experience it is not practical. The point of microgravity is to get crystals in a convection free environment, but this can be achieved by combining gels and caplillarys (check doi: 10.1016/S0022-0248(02)01837-7 and doi: 10.1107/S0021889801003697). If you decide to try this approach, search the literature for Gel Acupuncture Method (GAME).
Good luck.
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