On 6/2/2017 1:42 PM, wtempel wrote:
> Hello all,
> crystals with high solvent content tend to diffract poorly, at least
> according to intuition. Several years ago we solved a structure
> <http://www.rcsb.org/pdb/explore/explore.do?structureId=2h58> that
> appeared to buck that trend with a solvent content of ≈0.8 and
> resolution beyond 2 Å, per merging statistics and visibility of spots on
> diffraction images.
> I would welcome my colleagues’ opinions as to why I might observe the
> following:
>
>  1. Paired refinement (similar to Fig. 1 in Karplus&Diederichs
>     <http://doi.org10.1126/science.1218231>) indicates that adding any
>     higher resolution data beyond 3.4 Å, the lowest high resolution
>     cut-off limit I tried, does not improve R-factors at the common
>     lower resolution cutoff. Yes, diffraction is anisotropic in this
>     case, but seemingly not to that extent. I hesitate to “throw out”
>     all data beyond 3.4 Å, or whatever lower resolution cut-off I might try.
>  2. The Fo-Fc map, when countoured at ± 3 rmsd, includes many more
>     (uninterpretable) features than I would expect after refinement to
>     residuals in the mid-to-lower twenties. For expected map appearance,
>     I had to crank up the coutour level to > 5 rmsd, like in the
>     attached screenshot of the ADP·Mg^++ omit map.

   This is one of the prime examples of the failure of describing
contour levels in terms of "sigma".  First, the number you are using is
not a "standard deviation" or any other measure of the error level of
the map but is simply the rms value of the map.  If you calculate the
rms of a difference map where 80% of the unit cell is bulk solvent, and
therefore flat, you will, of course, get a much smaller number than if
the unit cell contained 80% protein with all the the expected difference
map features that come from a model with an R value of ~20%.  Then when
you contour at three times this absurdly small number you will see all
sorts of features you are not used to seeing.  Selecting a contour level
based on the e/A^3 is much less sensitive to the amount of solvent in
the crystal is gives much more consistent results.

Dale Tronrud
>
> Could these observations be linked to the high solvent content? (1) A
> high solvent content structure has a higher-than-average
> observation-to-parameter ratio, sufficiently high even when limited to
> stronger, low-resolution reflections? (2) Map normalization may not be
> attuned to such high solvent content?
> I am interested in analyzing the automated decision-making of the
> PDB-REDO of this entry <http://www.cmbi.ru.nl/pdb_redo/h5/2h58>, such as
> paired refinement results and selection of ADP model. Should I find this
> information in the “All files (compressed)” archive
> <http://www.cmbi.ru.nl/pdb_redo/cgi-bin/zipper.pl?id=2h58>? The “fully
> optimized structure’
> <http://www.cmbi.ru.nl/pdb_redo/h5/2h58/2h58_final.pdb> shows |ANISOU|
> cards and |NUMBER OF TLS GROUPS : NULL|. Does this mean that individual

> ADPs have been refined anisotropically?
> Looking forward to your insights,
> Wolfram Tempel
>
> ​