Hello Pisapia, I will start off by saying that my experience with X-ray crystallography and protein crystallization is limited. Soaking is a popular means of getting a co-crystal structure if co-crystallization does not work. Could you provide more information as to the identity of the protein and ligand(s)? What is the molecular weight of your protein? What is the function of the protein? If it is an enzyme, perhaps it would be a good idea to see if it is still active in the crystallization buffer (i.e. a functional readout that tells you if the enzyme can still convert, and therefore bind, the ligand). Is there a catalytically inactive mutant you could use that can still bind but not convert the ligand and, if so, perhaps that would be more successful for co-crystallization efforts. Cryo-EM would also be an excellent option, depending on the protein’s size, if you have access to an instrument. Good luck!

Jamison Drew Law thank you for your answer, it's simpler than that. It's a viral capsid protein that needs to recognise sugars to adhere on the host cell. My protein size is about 70 KDa and the ligand is about 1KDa. The question is more on the soaking tecnique to get the ligand into the binding site considering an affinity in the mM range.

Pisapia, see the following publication: https://febs.onlinelibrary.wiley.com/doi/10.1002/2211-5463.13913. I hate to give a “check the literature” answer, but I am sure others have co-crystallized viral capsid proteins with their respective ligands. You may be able to glean useful information from such publications, but the soaking condition that will work for you will need to be empirically determined. Sorry that I could not provide a more useful response, others may be able to assist you better. Good luck!

Pisapia Luca Would you happen to know if the binding of the ligand disrupts the crystal contacts? Also to what kind of solution is the ligand solubilized in?

Since you are not getting crystals while co-crystallizing, it could be that something in the solution is inhibiting nucleation, or the ligand is disturbing the crystals formation by interfering with the crystals contacts.

Talking about concentration, its all relative to the experiment and the soaking compound. 50mM is not "high", but I would recommend doing is multiple time point of soaking, starting from few minutes and going up from there (15, 30, 45, 120, etc). Observe how the crystals look in the soaking solution. If they become malformed, its either much of a shock for the crystals or the compound is disrupting the packing.

You can alternatively try less concentration for longer time.

For co-crystallization experiment, could you try to first add the ligand to the crystallization plate and let to dry. Then add your protein/precipitant ontop of the dried drop. This can sometimes help with co-crystallization. Although, my experience is with smaller molecules.

Another thing you could try is to do microseeding experiment with co-crystallization with the ligand. Take your best looking drop with crystals, and resuspend them to the precipitant solution before crushing them(either by vortexing or harsh pipetting). One drop for some 10-20µl of motherliquid. You can then make 1/10, 1/50 or so dilutions from this. Add the microseed solution to your co-crystallization drop. For example if you have a 300nl drop(protein+precipitant+ligand), you could add 20µl of the diluted microseed solution.

Hope you find any of this helpful.

Regards,

Mikko Hynönen

PS. Article by @Jamison Drew Law