As I have purified my protein and set crystallization screen and I tried various way to optimize the crystal quality, such as seeding, changing pH, PEG concentration, salt concentration and protein concentration and also tried two temperatures 4 degree and 20 degree. I am using the reservoir volume of 0.5 ml, but still did not get a crystal with nice morphology. Does anyone have any idea to improve the quality of crystal? Thanks in advance.
If you have tried a large number of conditions and your protein is clean and homogenous in solution and you still get no diffracting crystals I would suggest to change your expresiion construct. Remove flexible regions, change or remove tags, reduce tails exceeding folded domains. Having multiple constructs is often the key for successful crystallization of a protein.
Also you may require a cofactor or substrate to lock you protein in a stable conformation.
maybe you must test other crystallization methods like Micro-batch, FID, setting drop...
Growing crystals is more of an art than a science and luck is a major factor!
If you have tried a large number of conditions and your protein is clean and homogenous in solution and you still get no diffracting crystals I would suggest to change your expresiion construct. Remove flexible regions, change or remove tags, reduce tails exceeding folded domains. Having multiple constructs is often the key for successful crystallization of a protein.
Also you may require a cofactor or substrate to lock you protein in a stable conformation.
Dear,
Beside trying trial and error mode...
You should surely go ahead using co-factor/inhiitor to stabilize the protein and I am sure you will definitely get good quality crystals soon..
Cheers :)
As already mentioned, there are many things you can try. To improve the crystals in the condition you already have, you can try additive screen or set up with a ligand, if there is any, as already mentioned. If this doesn't help, you can methylate your protein and screen again or try different construct or truncation etc.
Jens had a really good advice, change your constructs. Actually, you want to start with a number of constructs you can screen in parallel. A good crystal construct will quite often crystallize in several conditions.
Of course you need to have constructs that contain all features you want to observe and not end up with an crystal artifact of your structure. I advise careful design and selection of expression constructs at the begin of every project. It is totally worth spending a significant amount of time on construct selection.
If you have crystals you can try do anything you want to improve them. Anything that gives you better crystals is legal, just document it.
There is no a priori way to tell what will help, you need to keep trying as many things you can think of and always permute back to cloning. Matrix microseeding seems to be a reasonable strategy for the initial screen of new constructs since you already have some kind of crystals.
In the end you typically end up with a fairly easy procedure to grow crystals, you just need to invest the time to figure out the protocol.
Hi Prem
Stefan has mentioned the most important set of techniques for this situation, matrix microseeding.
You should make a seed-stock from the crystals that you have and add them to a couple of RANDOM screen.
This approach is very likely to give you diffracting crystals if your protein is capable of generating them. The reason is that many conditions in the typical screen are in the metastable zone of the proteins phase diagram - i.e. there is a nucleation problem.
Google rMMS microseeding, or MMS microseeding.
Good luck with the project
Patrick
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