Hi Katherine
I'm regularly struggling with inhibitors that bound at non-canonical sites and
bind there with different conformations. So the situation in these
complex-structures could be a bit similar to your situation. In our case, the
inhibitors bind on the non-canonical site with roughly Kd 5-20mM, causing their
density to really wash out to an amount that parts of the molecule are no
longer resolved due to the absence of deep binding pockets. I know the behavior
you describe, namely that the molecule drifts over the epitope without creating
difference density at the new position.
Sadly, I don't have a solution that would help you out for sure, but here are
three tips:
- looking at the DS, I would assume that you have significant information
beyond 1.9A (completeness is still 94% and CC1/2 way over 60%. How much more
information can you bring into refinement? It took me a 1.1A DS to finally
resolve the proper orientation of a weakly, non-canonically bound inhibitor, so
it might be a good idea to extend the DS?
- try fem maps, might well be that they bring up important details to place the
molecule (however, in refinement, the ligand will drift off again...)
- Use an alternate method to track down weak interactions. If I cannot resolve
a complex satisfactorily, I switch to HSQC, which is much more sensible for Kds
in the mM range. Just use it to confirm that the ligand is actually binding
where you see the density (you can determine even Kds with that method)
Best,
Matthias
Dr. Matthias Barone
AG Kuehne, Rational Drug Design
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284
________________________________
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Katherine Lim
<katherine....@research.uwa.edu.au>
Sent: Friday, December 20, 2019 5:57:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Potential weak binding ligand in the active site
Hi all,
I apologise in advance for the long post. I am working on solving a structure
that looks like it could have a ligand bound in the active site. My data was
obtained from a crystal of just the soluble domain of my protein that had been
soaked overnight in the ligand solution. The apo crystal structure is already
known and so I have solved my structure using phaser MR. I have attached the
Aimless report output of my structure at the end of this email. The current R
values I have after refinement and adding in all the waters are Rfree: 0.2413
and Rwork: 0.1897. I can clearly see green density that is much larger (only
disappears when I contour the Fo-Fc map to about 6 A) than in my control
crystal that had been soaked in the same concentration of just solvent (I had
used DMF).
I am struggling to add in my ligand as it doesn't seem like the entire ligand
can fit in the green density. We think it may be because we have only used the
soluble domain and so the ligand isn't held very securely since the
transmembrane domain is missing. I have been trying to fit in smaller sections
of it and doing an occupancy refinement. So far I have been able to get part of
the ligand in with an occupancy of about 0.6 but after the refinement run,
phenix.refine seems to move this part of the ligand slightly out of the area
where I had tried to fit it into the green density. Interestingly, there isn't
a big red density in the area that this ligand section has moved to. I would
appreciate any advice on how I should proceed with trying to figure out if I
have tried the correct section of the ligand and the kind of refinement
settings to use with a weak binder.
Space Group P212121
Unit cell abc 84.76, 89.84, 91.55
unit cell alpha beta gamma 90, 90, 90
OVERALL LOW RES HIGH RES
Low res limit 45.78 45.78 1.94
High res limit 1.9 9.11 1.9
Rmerge 0.234 0.052 1.684
Rmeas 0.244 0.054 1.768
Rpim 0.069 0.016 0.527
Total # observations 663073 6282 36851
Total # unique 55174 579 3359
I/sigma 7.5 27.9 1.4
CC ½ 0.997 0.999 0.747
Completeness %
99 98.7 94.7
Multiplicity 12 10.8 11
Katherine Lim
PhD Candidate
School of Biomedical Sciences; School of Molecular Sciences
Marshall Centre for Infectious Disease Research and Training
The University of Western Australia
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