Naveed,
You mention:
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 you induce structural changes on ligand binding, lattice forces that
stabilize
one particular conformation may be able to fight against ligand binding.
The result
is not always absence of ligand. You will get a statistical sampling of
various viable solutions
but the answer in not satisfactory. You should try co-crystallization
instead of soaking!
For hydrophobic ligands the problem is different. (I assume that the
acidic tail
gives your ligand excellent water solubility so I will not mention what to
do in that case).
Enrico.
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Naveed A Nadvi
> Sent: Tuesday, April 24, 2012 12:02 AM
> To: [log in to unmask]
> 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.
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: [log in to unmask] Fax: 33 (0)1 69 08 90 71
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