Dear Naveed, >From your description, I get the impression that indeed you have a partially >bound inhibitor.
However, I do have some comments: At 1.7 Å, you should refine a group-occupancy for your inhibitor. With a partially bound inhibitor, the density in your active site will be a superposition of bound and unbound conformations and you should look if you could model both using alternative conformations and if that better explains the electron density maps. The proof of the pudding is the electron density: Does the electron density of your inhibitor after refinement look convincing? Many inhibitors are in fact slow substrates and still get converted if you incubate them long enough with the enzyme. If the inhibitor is expected to react covalently with the protein and the covalent cofactor is not covalently bound any more to the protein, this looks to me like you have incubated too long. If you did not try already, I would really recommend shorter soaks (1 hour, 1 day). Another thing to consider is to add fresh inhibitor as concentrated as possible prior to freezing. Especially if you do not add enough inhibitor to your cryprotectant solution, you may loose your inhibitor in seconds. Best regards, Herman -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 6:02 AM To: [email protected] Subject: [ccp4bb] Criteria for Ligand fitting Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi.
