Hi everyone
My protein has a molecular weight of 40 kDa. I have been trying to crystallize my protein and has not been able to observe any crystal but only precipitate in any screen. I have used hemp tons crystal screens 1 and 2. Where I might be going wrong?
Hi Ravindra,
First of all, I think the molecular weight of your target protein is suitable for crystallization, and because I don’t know its specific properties, I can only give you some basic suggestions.
1. Try as many screening conditions as possible, after all, crystallization is a matter of luck. According to your description, it seems that you have only screened the conditions of two kits, which is obviously not enough.
2. Precipitation has been observed in the results of your previous experiments, and I think it is a good omen because I think the crystal is regular precipitation.
3. Understand the properties of your target protein, and many factors can affect protein crystallization, such as the PH, concentration of the buffer, temperature, or other details of the operation. Moreover, you must ensure that the purified protein free of aggregation.
4. In my experience, you can try the hanging drop method (24-well plate), the nucleation rate of which is slower than that of the sitting drop method, but the crystals formed are larger and more regular.
5. The current expression and purification conditions may not guarantee that your protein used for crystallization is in a natural state, so it is recommended that you try to build other constructs or utilize other expression systems.
6. If none of the above attempts can solve your problem, it is recommended that you consider other structural biology methods that don’t require crystallization. https://www.creative-biostructure.com/comparison-of-crystallography-nmr-and-em_6.htm
Good luck.
Hi Ravindra,
Molecular weight is not the only parameter characterizing your protein.
I would recommend to study your protein more carefully.
1. I would recommend to run the expasy protparam https://web.expasy.org/protparam/
Check pI of your protein. Usually, precipitation happened when during the experiment the surface charge of the protein is neutralized completely, and that may happened when pH during the crystallization experiment will match pI of your protein. While you would like to drop the surface charge, you have to prevent total discharge. We are using Tris buffer with pH 8.3 for most of our acidic proteins, and pH 7.5 for our basic proteins.
2. I would recommend to run XtalPred
http://xtalpred.godziklab.org/XtalPred-cgi/xtal.pl
It will give you some ideas about how easy would be to crystalize your construct. It will check if there are signal peptides, or trans membrane fragments. If they are present in your protein, to increase the chances.of your protein to be crystalized, you have to remove them, and think about different construct.
3. Presence of precipitation could be related to the fact that you are not reaching supersaturation. Check if you can increase the concentration of your protein solution.
4. Very important part of your experiments is the state of your protein.
Did you set up your crystallization plates with fresh protein? If your protein was frozen, did you. properly thaw it? Some ideas about freezing and thawing... When you freeze your protein in tubes, you use liquid nitrogen, this makes your transition from liquid to solid state very fast. The same idea is behind thawing, you have to do it fast in your hand, just hold them in your hand and periodically shake tubes. This way you will avoid local high concentrations of your protein in the tube, and possible precipitation or denaturation.
5.Is your protein an enzyme ? Does it require any metals? If it is enzyme, are there available inhibitors? You have to think about what could make your protein "happy". This will help you to reach supersaturated state by increasing the concentration of your protein.
6. You can control the speed of the crystallization experiment.
In smaller drops processes are faster. In many plates the level of the well solution (or amount of air in the compartment) effects on the speed of crystallization - lower the solution level (large amount of air) will slow the crystallization.
7. In crystallization experiments the best reproducible results are when your precipitation (well) solution has all the additives from your protein solution. For example, your protein solution has 0.5M Sodium chloride, I would recommend to keep the same concentration of the salt in the well solution (adding into well solution). Ideally, your well solution has to have all components except your protein.
I hope this helps.
Good luck!
George.
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