Dear Sharifah, Was no protease inhibitor used during crystallization, or during the whole purification process? E.g. when a protease inhibitor cocktail was added during cell-lysis, a protease inhibitor may irreversibly have modified your serine.
If your protein is a protease, it may have reacted with a random peptide during purification and the modified protein may have selectively crystallized. In this case I would try to fit a few Ala residues as an acyl-intermediate, or even as a tetrahedral intermediate as the electron density seems to suggest, refine and see what side chains would fit best. You may also try mass-spec to get some information on the nature of your modification. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von sharifah nur hidayah syed mazlan Gesendet: Samstag, 4. Februar 2017 16:03 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Unknown blob extended from catalytic serine residue Dear All, I am working on a structure with an unknown blob extended from the gamma O of the catalytic serine residue. The resolution of the dataset is 1.38 A. I have no idea whether the residue is modified or the blob belongs to other molecule. The protein was expressed in Rosettagami (DE3), purified using Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange chromatography). The crystallization formulation used contain 15% PEG 8000, 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No protease inhibitor was used (eg: PMSF) I have tried to fit in diethylene glycol as shown in one of the attached figures, but as observed, it is not really fit and the molecule is in incorrect conformation. Kindly help me with this matter. Thanks and regards, Sharifah Nur Hidayah Universiti Putra Malaysia, Malaysia
