Thank you for your responses. Actually, the data are very complete: 99.6% total and 99.2% in the high resolution shell. Only 2% of data were used for cross validation because there were a large number of reflections.
I had calculated omit maps. At first when I discovered the presence of the lower occupancy form, I initially only had the modified ligand in the map, but then using phases from the model including it, noticed extra density explainable by the unmodified ligand. Afterward, however, I calculated omit maps, removing all ligands. The mFo-DFc omit map does indeed show features of the unmodified (lower occupancy) ligand even when omitting all ligands in the model. However, the 2mFo-Dfc omit map shows the lower occupancy form better than the mFo-Dfc omit map. Interesting. Gregg ******************************************* Gregg Crichlow Dept. of Pharmacology Yale University P.O. Box 208066 New Haven, CT 06520-8066 ******************************************* On 3/27/12 4:48 PM, "Ethan Merritt" <[email protected]> wrote: > [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 > >
