Re: [ccp4bb] Alternating positive and negative density
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
Re: [ccp4bb] Alternating positive and negative density
that should have been sort -gk5 XDS_ASCII.HKL | more (I should have tested before posting!) best, Kay On Tue, 25 Jun 2013 09:16:52 +0100, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: ... 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.
Re: [ccp4bb] Alternating positive and negative density
-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-
Re: [ccp4bb] Alternating positive and negative density
Based on eye-balling your map it looks to me that your grid spacing is about 0.5 A. The wavelength of your ripple is 4 grid spacings, and the ripple is right along the x axis. My guess is that you have a rogue reflection with index of h00 where h is about 2 A resolution. How you are getting this in multiple data sets is a mystery to me, but I would concentrate on finding that reflection and figuring out why it is anomalously large. Start with the Fourier coefficients that went into calculating this map to find the exact value of h causing the problem and then track that reflection back through your Fcalc's and Fobs's. Dale Tronrud On 06/23/2013 09:57 PM, 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 -- Peter Randolph PhD Candidate Mura Laboratory Department of Chemistry University of Virginia (434)924.7979
Re: [ccp4bb] Alternating positive and negative density
Hi Tim,
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.
Fully agree. I guess just typing them as 2mFo-DFc and mFo-DFc will solve
this particular confusion. Further on this:
- showing a map without specifying a contouring cutoff level (which also
provides information about how this map was scaled: by standard deviation,
volume or else) isn't very informative;
- was estimate of F000 included?
- which phases were used (model, Pc, or combined model+experimental, Pcomb);
- how phases were used. Example: {mcomb*Fo, Pcomb} - {DFc, Pc} vs {mFo-DFc,
Pc} and other flavors of this;
- are Fc structure factors calculated from atomic model only or it is
actually total model structure factor Fmodel = overall_scale * (Fcalc_atoms
+ Fbulk_solvent)?
All the best,
Pavel
