Dear All;
In response to my "Anisotropic Diffraction In Refinement", which asked
for suggestions for how best to proceed with refinement with an
anisotropic data set, I received a large number of responses which
overwhelmingly suggested using the UCLA Anisotropy Server
(<http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/>).
The Anisotripy Server treats scaled/truncated data sets (I used Scala
and the old Truncate program). Fo and SigFo are analyzed with respect
to resolution in three dimensions and the data treated in three steps:
1) An elliptical resolution boundary is determined and applied.
2) A purely anisotropic B-factor is applied to the Fo and SigFo data
to cause the data in all directions to fall off equally.
3) A negative isotropic B-factor is then applied to the structure
factors to force the fall-off in the strongest direction to match that
of the original data, effectively meaning that the data are not scaled
to the mean but the weaker data are scaled up to match the strongest
data.
Application of a elliptical resolution boundary is justified because
the resolution boundary from common integration programs (Denzo and
Mosflm for example) is spherical where diffraction for anisotropic
data is ellipsoidal. A spherical boundary would result in the
inclusion of numerous poorly measured reflections in the higher
resolution shells which effectively makes these data more noisy.
Imposing an ellipsoidal resolution boundary is equivalent to removing
noise from the higher resolution bins and is simply the anisotropic
equivalent of the normal resolution limit truncation.
However, I was confused by the second and third steps. The second
step of application of anisotropic scale factors is appropriate if the
refinement program does not include anisotropic scaling in its
calculation of Fc, however modern refinement programs do this. Pavel
Afonine touched on this in his CCP4BB general posting in response to
my original posting where he noted that "anisotropic scale factor[s]
that [are] part of the total structure factor take care of this"
(<https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0909&L=CCP4BB&T=0&F=&S=&P=8362>).
For the third step, applying a negative isotropic B-factor to modify
the Fo is equivalent to sharpening the peaks in your maps and this can
be useful. However, applying the correction to Fo will also result in
an inappropriate decrease in the average temperature factor of the
resulting model. Since B-factors are used as a measure of the
coordinate error of an atom, modifying your Fo means these low B
factors will tend to confuse the users of that model
into thinking its quality is better than it really is. If a sharper
map makes identification of model errors easier, the map can be
sharpened when it is calculated, without affecting the parameters in
the PDB file. The latest versions of Coot, for example, allows you to
sharpen any map that it calculates.
I brought these points to the attention of the Anisotropy Server
director (Michael Sawaya), who is now working to provide an option to
omit steps 2 and 3 for users who do not what their structure factors
modified.
My thanks to everyone who responded to my original question, and to
Dale Tronrud and Michael Sawaya in particular for valuable discussion.
