I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification?
thank you for suggestion. Dhiraj
