[Snipped from the full message, which is appended below]

The program that kept showing me two forms bound was not

substituting Fcalc for unobserved reflections.  So, I turned on the option

to substitute Fcalc, and the minor form disappeared � the density looked

like it did in the second program.  I figured the density that reveals the

two forms must be correct being that it would be a big coincidence for

artifactual density to appear that just so happens to fit perfectly our

added (unmodified)ligand at 1.55 A.  So, I suppose, being that the occupancy

of the major form is so much higher, by substituting unobserved reflections

with Fcalc, the major form is being overemphasized, and the minor form

becomes invisible.

A weighted difference map (mFo - DFc) that does not include the ligand

atoms in Fc at all would be a better guide.  It would not be biased by

the current ligand model [or at least much less biased] and it certainly

would not be sensitive to the modelled occupancies since these atoms

would not be contributing to Fc at all regardless of occupancy.

It is in general more convincing to show difference density from

an Fo-Fc map with ligands omitted from Fc than it is to show density

from some variant of 2Fo-Fc with ligands included in Fc.

How complete is your data set?  

Are you trying to deal with more than a few per cent of missing reflections?

If it is only the highest resolution shell that has poor completeness,

have you tried truncating the map calculation to a shell that is complete?

Ethan

On Tuesday, March 27, 2012 01:06:16 pm Gregg Crichlow wrote:

Please excuse me for bringing up an old issue.  I have an interesting

example of a difference seen when DFc was substituted for missing

reflections versus when it wasn�t. Maybe others had this experience.  I had

a structure in which the electron density showed two �overlapping� ligands

bound in the same active site.  One was the ligand that was co-crystallized

with the protein.  The other was the same ligand but with an unintentional

modification (presumably due to radiation dose).  I was able to discern the

two forms in the electron density (1.55 A) being that they did not

completely overlap.  Based on occupancy refinement, the occupancies were

0.12 and 0.88 (unmodified and modified forms, respectively).  Then one time

I calculated the map using a second program, and the lower occupancy ligand

disappeared!  When I calculated maps in the first program, there were again

two forms visible.  I thought that the difference may be due to the

difference between substituting unobserved reflections with Fc (or rather

DFc because of sigma-A weighting) versus omitting them from the Fourier

transform.  The program that kept showing me two forms bound was not

substituting Fcalc for unobserved reflections.  So, I turned on the option

to substitute Fcalc, and the minor form disappeared � the density looked

like it did in the second program.  I figured the density that reveals the

two forms must be correct being that it would be a big coincidence for

artifactual density to appear that just so happens to fit perfectly our

added (unmodified)ligand at 1.55 A.  So, I suppose, being that the occupancy

of the major form is so much higher, by substituting unobserved reflections

with Fcalc, the major form is being overemphasized, and the minor form

becomes invisible.

                   There may be many cases in which substituting Fcalc (or

DFc) for missing reflections is beneficial. I don�t know the mathematical or

theoretical arguments behind it.  I�m not arguing for one way being

generally superior to the other, or for one program over another.  However,

this is one empirical example of it being advantageous not to make this

substitution.

                   When calculating experimentally phased maps, we multiply

our structure factors by a figure of merit to down-weight reflections with

less certain phases. Could one consider leaving missing reflections as zero

analogous to multiplying Fcalc by FOM = 0? (just asking � maybe this is

faulty logic.) Of course, this would be for the sake of the amplitude

instead of the phase in this case. If an intensity is not observed, we have

the ultimate uncertainty regarding its value.

Maybe some developers will want to use this structure and the corresponding

data to test DFc vs. �0� vs. DFc multiplied by a specific FOM only used for

the missing reflections, varying from 0 to 1.  Unfortunately, this structure

is not yet published (we needed to wait for other experiments to be

finished) so I cannot yet provide it or the structure factors. However, if

anyone is interested, feel free to contact me, and when it is published I

would be happy to let you know the PDB code, if you still want it.

*******************************************

Gregg Crichlow

Dept. of Pharmacology

Yale University

P.O. Box 208066

New Haven, CT 06520-8066

*******************************************

  

--

Ethan A Merritt

Biomolecular Structure Center,  K-428 Health Sciences Bldg

University of Washington, Seattle 98195-7742