[phenixbb] refinement of partly occupied water molecules?

[Nathaniel Echols]

On Thu, May 30, 2013 at 5:48 AM,  <krimmer at staff.uni-marburg.de> wrote:
> in some of my macromolecular crystal structures with resolutions between 1.1
> - 1.4 Å, mainly in the second solvation shell round positive Fo-Fc electron
> density blobs are detectable which show after assignment of a water molecule
> to these blob and subsequent refinement with Phenix refine a good-looking
> calculated 2Fo-Fc electron density (round shape, the 2Fo-Fc map appears at a
> contour level of σ = 1.0 - 1.4), but there also occurs a small negative
> Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The
> negative Fo-Fc electron density disappears if the occupancy of the water
> molecule is refined by Phenix or manually set lower than 100% occupancy.
> Therefore, I think these positions are partly occupied by water molecules,
> but I am not sure how I should handle it/how it is generally handled. I
> would be thankful for any advice and/or literature about this topic.

The only explicit advice I've seen is in the SHELX manual:

http://www.msg.ucsf.edu/local/programs/shelxl/ch_08.html

"It is relatively common practice in the refinement of macromolecular
structures to insert water molecules with partial occupancies at the
positions of difference electron density map peaks in order to reduce
the R-factor (another example of ' R-factor cosmetics'). Usually when
two different determinations of the same protein structure are
compared, only the most tightly bound waters, which usually have full
occupancies and smaller displacement parameters, are the same in each
structure. The refinement of partial occupancy factors for the solvent
atoms (in addition to their displacement parameters) is rarely
justified by Rfree, but sometimes the best Rfree value is obtained for
a model involving some water occupancies fixed at 1.0 and some at
0.5."

> My second question is concerning a density blob in perfect coordination
> distance to an indolyl group (perfectly located in the middle of the
> five-ring, with 3.4 Å distance to every of the five carbon atoms of the
> five-ring). What‘s the best way to find out which ion possibly occupies this
> position? I tried Na+ and Ca2+, but after refinement big negative Fo-Fc
> blobs appear in both cases (maybe the ion at this position is only partly
> occupied?). There is no anomalous signal detectable at this position (data
> collection wavelength was 0.91841 nm). After the refinement of a water
> molecule at this position, a positive Fo-Fc election density is still there.

The nightly builds have an option in phenix.refine to place ions
automatically based on a variety of criteria (still being tuned).
However, what you describe doesn't sound like any ion coordination
motif that I've read about.  Could you please send us a picture that
shows the density in context?  Some of the other readers with more
experience in "blobology" may recognize it.  [if our list server
complains about the size of the message, just ignore it, I'll approve
it for posting]

-Nat