Dear Akanksha,
When you re-do the occupancy refinement, don't include a b-factor
refinement along with the occupancy refinement. Do them separately. Do
one, then do the other, and then repeat until convergence.
If your B-factor values are either high or low, the computer will try to
adjust using occupancy. If your occupancy values are either high or
low, then the computer will try to adjust using B-factors. In the end
you want the best occupancy and b-factor estimates you can get by making
sure the refinement programs don't get trapped.
Once you have a good estimate, I suggest that you try a few different
initial occupancy values to make sure you are not trapped in a local
minimum. If you start off with a high occupancy, it should move back
down during the refinement. If you start off with a low occupancy, then
it should back up during the refinement. B-Factors should do the same
thing.
With a large ligand, I suspect that some of the waters will also have
alternate orientations. You also might look at the amino acids that
bind to the ligand to see if there is any alternate conformations.
Best
Steven Herron
On 3/3/2022 8:51 AM, Akanksha Tomar wrote:
Thank you for the suggestions
I will try again by setting the occupancy of the entire ligand to the
single average occupancy and re-do the refinement with Buster, Phenix
refine and Remac5 with full positional and B-factor refinement and
check the B-factor of the neighbouring residues.
The ligand is a 30 atom containing molecule binding at a shallow
solvent-exposed site.
On Thu, 3 Mar 2022 at 20:38, Wim Burmeister <wim.burmeis...@ibs.fr> wrote:
Hello,
at 2.1 A resolution, atomic temperature factors and occupancy are
strongly correlated. So you have to be very careful with the results.
So the best is just to set the inhibitor to the average occupancy
and then to include it into a full positional and B-factor
refinement. You can check whether the result is coherent by
comparing the B-factors of the ligand and of the atoms, which are
in contact with it. If this is not the case, you may want to
adjust the occupancy manually. As ther are also solvent atoms at
the ligand positions, when it is not bound, there is another
source of inaccuracy and theoretically you would have to model the
site with the solvent and an occupancy 1-q and the ligand with an
occupancy q as alternate structures. But nobody does that and it
is not really required.
Best
Wim
------------------------------------------------------------------------
*De: *"Akanksha Tomar" <akankshat...@gmail.com>
*À: *"CCP4BB" <CCP4BB@JISCMAIL.AC.UK>
*Envoyé: *Jeudi 3 Mars 2022 15:15:07
*Objet: *[ccp4bb] Ligand occupancy refinement
Hi everyone,
I am trying to refine the occupancy of a bound ligand. After
fixing the protein model and water I fitted the ligand into it.
Currently, I am using Phenix Refine with occupancy refinement for
individual atoms switched on. After the refinement, the overall
occupancy of the ligand is 0.7 and the RSCC value is 0.86. The
resolution of the structure is 2.1 Å.
Now the problem is that the program has assigned different
occupancies to different atoms of the ligand. For some cases, it
has assigned 0 occupancies to atoms for which there is a clear
positive peak.
Why it has been done so and is it acceptable?
Any help would be greatly appreciated.
Thank you.
--
Best Regards,
Akanksha Tomar
Pre-Doctoral Fellow,
C\o Dr. Arockiasamy Arulandu,
Membrane Protein Biology Group,
International Center for Genetic Engineering and Biotechnology,
New Delhi, India
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--
*Wim Burmeister*
Professor
Institut de Biologie Structurale (IBS) CIBB
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