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:[email protected]] On Behalf Of
Naveed A Nadvi
Sent: Tuesday, April 24, 2012 12:02 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.
--
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: [email protected] Fax: 33 (0)1 69 08 90 71
