Hi Kyle

You don't say what protocol you're using for the 10-fold NCS restraints (if
any).  If the NCS restraints are too tight compared with the differences
between NCS copies this will push up the protein B factors (and the R
values) to compensate.  So the problem may not be that the water B factors
are too low, rather the protein B factors are too high due to the overly
tight NCS restraints.  Also as Rafael asked how does the Wilson B factor
compare with the averages for the protein and waters - that might give a
clue.

Cheers

-- Ian


On Tue, 6 Jan 2026 at 14:48, Kyle Gregory <
[email protected]> wrote:

> Dear all,
>
> I have a 2.6 angstrom structure, which has 10 molecules in the ASU. I have
> modelled approx 200 water molecules. They fit spherical density nicely and
> make suitable contacts with the protein. The only problem is nearly every
> single water molecule has a lower B-factor than the protein atoms they
> coordinate. E.g protein atoms could be 30/40 while water is 16 angstrom^2.
>
> Does anyone have a suggestion as to why, and whether there are some things
> I should be trouble shooting?
>
> Kind regards,
> Kyle
>
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