Hi everyone!
I'm a freshman in the crystallographic field.
Now I have a crystal that have been confirmed as protein, and it looks like a dot without clear edge.
I have tried to optimize the crystallographic condition,like the concentration of precipitant and the pH range of buffer.
but the resolutioin is only 5 Å using X-ray diffraction。
So could you please share your experience to improve the resolution of protein crystal?
Thanks, sincerely.
A resolution of 5% is too low to yield meaningful 3D structure. Improve your crystal by making sure your protein is as pure as possible and as concentrated as possible before trying to crystallize it. IOW, invest more time in crystallization - it will pay off when diffraction time comes. And try to grow your crystal as large as possible so that a single crystal can withstand the x-ray beam long enough to yield a complete data set (otherwise you will need to collect diffraction data from several small crystals just to obtain a complete data set, and merging several diffraction data sets from several crystals is quite a problematic process). Also, try not to move around your crystal tray too often (e.g., looking under the microscope twice a day) as this movements can cause lattice disorder ("mosaicity") in the crystal, which will lower its resolution under diffraction. HTH.
You may simply want to attempt further (wide) screening to find additional crystal forms. I would also advise you to do additive screening (e.g. using the Silver Bullets additive screens). I have had a lot of success improving resolution via additives.
https://www.hamptonresearch.com/product_detail.aspx?cid=30&sid=179&pid=562
In situ proteolysis (addition of trace amounts of protease to your xtallisation experiment) has also been reported to be successful at finding new crystal forms with improved diffraction quality. You can read up on it here:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366506/
Another fast and efficient way of potentially improving your resolution is protein surface engineering in the form of lysine methylation (basically to reduce surface entropy). You can read up on this here:
https://www.ncbi.nlm.nih.gov/pubmed/17098187
The above methods won't require you to go back to cloning. However, stepping back to reflect on your construct is often best. You may want to introduce mutations that improve crystallisability through surface entropy reduction. Have a go at this server if you want:
http://services.mbi.ucla.edu/SER/
Finally, use disorder prediction software to check if there are flexible regions on your protein which you can possibly remove.
Happy screening!
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