I'm trying to get a protein crystal with a new compounds and there was reported x-ray structure of this protein. However, when I follow the crystallization condition of related paper, I get very thin crystal and there're too many small crystals so that no enough big crystals grow. The condition was 0.1 m sodium acetate, pH 4.2–4.6, 1.6–1.8m sodium formate. How can I optimize this conditions?
Dear Minghui, many thanks for your very interesting technical question. Your crystals already look quite nice but, as you said, they are perhaps a bit too thin for X-ray diffraction. In this context please have a look at the following potentially useful articles which might help you in your analysis:
Optimization of crystallization conditions for biological macromolecules
Article Optimization of crystallization conditions for biological ma...
and
Protein crystallization: virtual screening and optimization
Article Protein crystallization: Virtual screening and optimization
Also please see the following Open Access guide:
Critical factors for successful protein crystallization
(cf. attached pdf file)
I also suggest that you check the "Publications" section of RG to find and access other relevant papers in your field. For example, just search for the term "protein crystallization" and then click on "Publications". This will provide you with a long list of interesting papers which have been posted on RG, many of them even as public full texts:
https://www.researchgate.net/search.Search.html?type=publication&query=protein%20crystallization
Good luck with your work and best wishes!
Dear Minghui Liu , I join to Frank T. Edelmann both about the nice crystals you already have and the great references and advice provided by him. Just let me add that the very first problem is to get crystals, specially with proteins. Your conditions were successful to obtain nice crystals, but too many and then small. A common procedure to obtain bigger crystals would be to use some of these small crystals as seeds in a new batch with the same conditions or some new ones. The presence of some crystal seeds will - in general- promote the growth of these seeds.
Hope it helps. Good luck with your crystals!
Dear Minghui Liu ,
I second the excellent recommendations by the distinct colleagues Frank T. Edelmann and Manuel Gómez .
Let me just add the following link (no commercial promotion intended!), where you can find interesting references for the "random microseed matrix screening" protocol:
https://www.douglas.co.uk/mms.htm
In my case, this has allowed me to go from thin plates diffracting to 8-10 A, to 3D crystals diffracting to 1.95 A (manuscript in preparation). If you have access to a crystallisation robot the work is facilitated. However, you can do it by hand (although more troublesome and using more protein).
Best of luck for your future endeavours!
HTH, best,
Jose
Hi. I hope the following article might help you very much:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231845/
Hi Minghui,
If you want to accelerate nucleation, you can try inoculating the crystals from the sitting drops into the droplets of the hanging drops to promote nucleation.
Good luck!
Crystal seeding might help you.
https://hamptonresearch.com/uploads/cg_pdf/CG101_Seeding_2020.pdf
Centrifuging a protein sample through a 0.20 micron spin filter does a good job of removing aggregates and junk from the protein preparation. In the case of lysozyme and probably other proteins it is the difference between a few ginormous crystals in a droplet and a lot of smaller crystals.
By the way, I study an enzyme which is fully inactivated at pH 5.8. It doesn't bind any substrates or inhibitors at that pH. Just crystalizes nicely by excluding all its substrates it loves at physiological pH. Maybe it is worth testing if you enzyme demonstrates nonzero catalytic activity at acidic pH.
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