Hi Peter, my estimate of the distance between the peaks would be based on bond distances and about twice that of Dale, but I agree with his general conclusion. Either a reflection (roughly in the h 0 0 direction) is stronger than it should be (ice?), or a very strong reflection was considered as outlier and removed (in the CORRECT step). I would try two things: a) to confirm the general conclusion, I would compare the "filled" (electron_density_maps.map_coefficients.fill_missing_fobs=True) and the default "non-filled" maps from phenix.refine. The "filled" maps should show the problem much less. (Caveat: I have not tried this) b) inspect the outliers in XDS_ASCII.HKL with something like sort -nk5 XDS_ASCII.HKL | more looking for observations which have negative sigma (i.e. are outliers) _and_ have high intensity _and_ have |h| >> |k| _and_ |h| >>|l| . If you find any, I would simply remove the negative sign with an editor, save the modified XDS_ASCII.HKL and re-run XDSCONV, finally obtaining a MTZ file that includes this reflection.
HTH, Kay On Mon, 24 Jun 2013 00:57:49 -0400, Peter Randolph <ps...@virginia.edu> wrote: >Short version: >Hi, I'm working on what should be a straightforward molecular replacement >problem (already solved protein in new space group), but my Fo-Fc map >contains a peculiar series of alternating positive and negative peaks of >difference density. I'm wondering if anyone has anyone seen this before? >Sample images are attached and more background is below. > >More background: >I had initially solved an *apo* structure of my protein (from previous >diffraction data in another crystal form), and more recently collected >diffraction data for crystals of the protein co-crystallized with potential >binding partners (small RNAs). All the datasets I've processed so far have >the same spacegroup (P2(1)2(1)2(1)) and cell dimensions as the apo >structure. > >I have tried two general approaches, both with the same initial steps of >indexing / integrating / scaling in XDS, converting to MTZ format without >R-free flags, then importing R-free-flags from the (previous) apo >structure's MTZ. I would then run "phenix.refine" for initial rigid-body >refinement using the apo-model and the new mtz to see if there were signs >of any new positive density corresponding to bound ligands. While the >2Fo-Fc map fits the apo protein 3D model perfectly, the Fo-Fc map shows >bands of alternating positive and negative density running throughout the >structure. What's odd is that these 'bands' appear to be systematic rather >than random (please see attached image), and aren't located anywhere that a >binding partner could bind, leading me to suspect they may be artefactual >(these bands actually run through the body of the protein, so one >possibility is that the b-strands are off-register by a multiple of a >peptide unit?). If I use the same mtz file and structural model, and >instead do molecular replacement with phaser, I see the same issue. I've >tried this workflow with a couple of datasets and using P222 as well as >P2(1)2(1)2(1), and each time I see the same issue of spurious(?) bands. Any >help or advice would be much appreciated, especially if anyone has seen >anything like this? > >Thanks a lot, >Peter Randolph > >-- >Peter Randolph >PhD Candidate >Mura Laboratory >Department of Chemistry >University of Virginia >(434)924.7979 >
