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CCP4BB  April 2012

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Subject:

Criteria for Ligand fitting

From:

Naveed A Nadvi <[log in to unmask]>

Reply-To:

Naveed A Nadvi <[log in to unmask]>

Date:

Tue, 24 Apr 2012 05:02:15 +0100

Content-Type:

text/plain

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text/plain (14 lines)

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.

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