Recently, I did crystallization screening of my protein after purification and few conditions produced crystals, some of which were large and confirmed to be protein crystals. However, I am not being able to produce the crystals in those conditions. I tried crystallization in those conditions using the reservoir solutions from the manufacturer as well as optimized solutions prepared by myself. However, none of the drops produced crystals. Could anyone please give me some suggestions?
Did you also use the same protein batch? sometimes even very small differences could change the output from outstanding crystals to nothing at all.
Try crushing some of the crystals you already have and using them as seeds for crystallizing more of the same protein under the same conditions.
Sometimes the exact conditions to grow a certain crystal are not very easy to reproduce. Just try several times trying and take on account every single variable (room temperature, humidity, vibrations, buffer conditions, drop volumes). Sometimes literally just a small particle of dust can be a nucleant agent for some crystals, so don´t be afraid of the negative results and just try and try again....and at the end you will be able to grow them again!
Good luck!
Providing that your are using the same batch of protein and that it was not altered by several cycles of freeze and thaw (i.e. ruling out the protein as the source of problems) I would pay special attention to:
- Composition of the reservoir: It is typical to store crystallization kits for long periods of time that lead to a change in the composition of the cocktail and some times, different kits with the same crystallization conditions yield different results (https://www.ncbi.nlm.nih.gov/pubmed/12657807). You should try grid screening from the results obtained with the kit.
- Drop: Make sure that you have not changed the geometry of the system. There is a clear correlation between volume of the drop and size of its surface (keep in mind that surface influences the rate of diffusion). It is also important to maintain constant the volume of reservoir since the space between the cover slide and the reservoir will be saturated by vapour. On the other hand the preparation of the drop is also important: the order of addition matters.
The temperature of the solutions also matters (http://www.nature.com/articles/srep07797)
If everything is under control and you do not get the expected crystals, depending on what your current results with the reservoir solution from the manufacturer I would focus:
If you get precipitation or small crystals: Your supersaturation is too high (is the temperature in you lab the same as when you succeeded?). You have to decrease the supersaturation of your system before setting your experiment by: i) dilution your protein (this is not a good idea), dilution of you reservoir (this is cheap and straightforward), change the protein:reservoir ration in the drop (this may be time consuming since you move the search area in the phase diagram)
Regardless of your results (clear solution or precipitation) I would use solution from the manufacturer and manipulate the ionic strength of the reservoir.
Good luck.
Hi Rakesh,
Javier gave you the most complete recommendations.
I have few more...
First of all you did not mention how long did it take for your crystal to nucleate and to grow from the time of crystallization plate setups.
1. If you got crystals less than in 1 month, then you should be able to reproduce your crystals. If your crystals started to grow after 1 month, there is still possibility to get crystals again, but you will not able to control the situation.
2. Your protein could be very sensitive to freezing. There are special rules how to freeze and how to thaw crystals. Both processes should be fast. That is why is a good practice to freeze protein solution in small tubes. The amount which you would use for one plate. In our lab that is 150 uL. When we place tubes in liquid nitrogen the freezing process is very fast.
To thaw your protein you have to be fast. The best way is to thaw in your hands. You shake the tubes all the time and control the process.
To find our if your protein is sensitive to freezing, would be to check your tubes before the crystallization. If you have precipitation, that would mean, that you have problems with freezing and thawing of your protein, or your protein does not like to be frozen.
The solution for this – a new purification or production of your protein.
In many cases, even if you did not get precipitation, some amount of your protein in solution after freezing and thawing will undergo some conformational changes or even denaturation. That would drop the effective concentration of the native protein in the solution, which was giving crystals when protein was "fresh".
Another question would be: What is your goal?
To repeat the crystallization conditions or to get crystals and solve the structure? If you are interested in the crystal structure, than look on each your crystallization as a new experiment. Do not expect to get crystals at the exact conditions. It is like waiting for your bus at the bus station. It may come few seconds later, it could be your bus number, but a different bus, or different driver... Most important is that it will come and take to your destination.
Good luck!!!
George.
Thank you, George Minasov and all others for your kind suggestions. I will use these suggestions and hope to get new crystals.
I faced this problem since from one year.I got the crystals after 4 months and diffracted 4A. However, i tried to reproduce the crystal, but was unable to reproduce it. I dont know the reason, but when i was checking the crystal plates under microscope, i was seeing precipitate inside the drop. i GRID it with different variables ,but could not reproduce it.My protein is stable at room temperature.Could i change the temperature parameter. please Could any one provide the suggestions.Your help will be highly appreciated.
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