Hi All, I want to confirm the binding of the substrate in the active site of the enzyme by soaking crystals of the apo-protein with the substrate. I generated a Fobs_lig - Fobs_apo difference electron density map. Inspection of the map did not show the characteristic electron density of the substrate. However, the difference electron density map (1Fo - 1Fc) generated after restrained refinement reveals the characteristic electron density of the substrate. I am scaling Fobs of both the ligand bound and apo proteins and using the phi_c from the apo to calculate Fobs_lig - Fobs_apo difference map. Can this problem arise due to loss of isomorphism in the crystals after soaking with the substrate? The c-axis dimension increased by ~10 angstroms after soaking while the a and b axises increased by less than an angstrom. Moreover, I need to do a full blown MR instead of rigid body replacement with the apo protein.
Thanks, Rajan.
