-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Hello Peter,
have you tried removing a few residues involved and (after a round of refinement) rebuild the area from scratch (a simple way to remove model bias)? How did you decide about the resolution cut-off of your data sets - could it be that there is noise and that it might be worth cutting a little more during integration? Although I agree the difference map seems to have systematic features that do not quite support this suggestion. A side note: you are most likely not looking at 2Fo-Fc and Fo-Fc maps, but a sigma-A weighted maps and sigma-A weighted difference maps. I think it is worth differentiating between these terms. Second note: thumbs up for the small size of your attachments. I think this is a good compromise between something quite impossible to describe and people who do not like large attachments. Regards, Tim On 06/24/2013 06:57 AM, Peter Randolph 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 > - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRyDLSUxlJ7aRr7hoRAvzCAKCER6EKt6GVsiXEpLx1GjYDNWKY/gCfRrKv LCo7f33FHgeevC9jo7m/kaw= =OjWN -----END PGP SIGNATURE-----