Interesting. One of the most helpful ways of ascertaining whether you do have a bound ligand (or, as Jay suggests, bound but modified ligand/protein) is, of course, to compare the electron density you see in the complex with the corresponding unliganded (let’s say apo) complex (in your case, the crystals before soaking).
One can calculate m|Fo(soak)|-m|Fo(apo)| maps (i.e. in reciprocal space). Separate maps can be generated using phases from both the soaked and apo structure, i.e. (m|Fo(soak)| -m|Fo(apo)|) exp–iphi(soak) and (m|Fo(soak)| -m|Fo(apo|)) exp –iphi(apo). These can be compared to check the level of phase bias in the maps. Of course omitting the ligand or active site waters may further reduce phase bias and there are other approaches to reducing phase bias that can be incorporated. To go a step further, you can even calculate a difference map, in real space, where you subtract the electron density of your (m|Fo(soak)| -m|Fo(apo)|) exp –iphi(apo) map from your (m|Fo(soak)| -m|Fo(apo)|)exp –iphi(soak) map; this will help show any phase bias. This exercises the mind when it comes to remembering what a positive peak and what a negative means (and exercised my mind when trying to describe these maps correctly in ascii - which I may well have got wrong!) Should the cells of your soaked and apo structures differ my more than a percent or so in dimensions or more than a degree or so in angles, you may be restricted to working solely in real space (a high Rmerge for Fo(apo) to Fo(soak) will tell you whether the structures are too isomorphous fro reciprocal space comparison – perhaps this is the case as you mention you used MR to solve the structure?). In this case, you can still generate an (m|Fo(apo)|) exp -iphi(apo) map and subtract it, in real space, from an (m|Fo(soak)|) exp i-phi(soak) map, effectively giving you a rho o(soak) - rho o(apo) map. It’s important to look at all these maps to get a clear picture of the differences your processed data show, as opposed to the differences which result from your two sets of phases. It’s worth noting, you should not expect to see strong peaks in these maps in positions where water molecules are displaced by your bound ligand. 1.7A should be plenty high enough resolution for this and all can be done easily in CCP4. Don't be put off by what seems a tedious set of maps to calculate, these can be quite instructional and generating a master mtz file containing h, k, l, fo(soak), fo(apo), fc(soak), fc(apo), sigf(soak), sigf(apo), phi(soak), phi(apo) will allow you to pick and choose maps as you see fit. Let us know what you find, Colin -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pandit, Jayvardhan Sent: 24 April 2012 14:56 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Criteria for Ligand fitting Naveed, From your description, it appears as if some covalent changes have occurred in the 2 weeks of soaking. Perhaps your inhibitor is getting turned over by the enzyme ? Also, if you don't have your inhibitor in the cryo, you could be soaking it out prior to data collection, which may explain the weak density. Good luck, Jay -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 12:02 AM To: CCP4BB@JISCMAIL.AC.UK 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.
