I'm working on a new class of transporters in bacteria. I optimized the protein yield to about 3 mg per liter of culture, therefore I can generate enough protein to play with. So far I have tried a range of different crystallization screens but still I don't have any crystals. Considering that membrane proteins have different requirements and specificities, any suggestions how to elevate the chance of crystallization of this transporter? Thanks
Hello! What detergent are you using? There are a number of good detergents to try. A good place to start would be a detergent with the smallest micelle that will still give a nice peak on size-exclusion in complex with your protein. Are you using a mosquito, or other nl drop robot? If not, the number of screens you can try is limited. There is a book by Therese Bergfors "seeds to crystals" it has a ton of strategy for developing protein crystals. Especially useful without a mosquito. Other things to try is truncate overly flexible regions and co-crystallization.Good Luck!
https://www.ncbi.nlm.nih.gov/pubmed/12718920
Hi Matthew, Thank you for your swift reply. I am using 2% DM. I haven't tried other detergents but since it gave me a good yield so far I'm sticking to it. Do you think I should still try other detergents? I have access to Gryphon crystallization robot. In each prep I try at least a few different screens.
I will try both truncation and co-crystallization soon. Thanks.
You can co-crystallize the transporter protein with the molecule that it normally transports. You will then be crystallizing the binary complex, which is usually more stable and compact than the apo-transporter ("empty" transporter), which can only make them crystallize more readily. Plus you will get more information from the crystal structure of the binary complex than from the apoprotein.
Hi Ali! I don't know how attached to DM you are, but generally, it is a good idea to try different detergents. Having the different types of detergent micelles facilitates different possible crystal contacts between micelles. There are only a few detergents that have resulted in the vast majority of structures, DDM, DM, b-OG, FC12 (I've not used). Besides this relatively small number your transporter may not be stable in them. This you test by size exclusion in the new detergent. Watch for precipitation, as you wouldn't inject a precipitating protein preparation onto a SEC column. Also 2.0% DM is way over the CMC of the detergent. This may interfere with proper crystal contacts. A rule of thumb is 10-20% above the CMC as the upper limit. If you can do the screens in nanoliters sometimes its good to optimize pH against one precipitant (PEG 1K). Then if you get any glassy ordered precipitate in your drops try seeding with a cat whisker as in the Bergfor book. Good luck membrane protein crystallography is a really hard project.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062488
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