Hi John, Thanks again for helping us! Regarding the map after placing missing residues and refinement: you are correct, both the negative and positive densities have gone away from the loop. Thanks, Renu
--------------------------------------------- Renu Kadirvelraj, Ph.D. Research Scientist A428, Biochemistry and Molecular Biology 120, East Green Street University of Georgia Athens, GA 30602 Tel: (706) 583 0303 ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of John Bacik <[email protected]> Sent: Friday, July 26, 2024 11:18 AM To: [email protected] <[email protected]> Subject: Re: [ccp4bb] Presence of negative density in Sim Omit map You don't often get email from [email protected]. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> [EXTERNAL SENDER - PROCEED CAUTIOUSLY] Hi Renu, how do the maps look (2Fo-Fc and Fo-Fc) after you run a standard refinement in Phenix with the missing residues in place? If the negative density disappears in the Fo-Fc then there is not necessarily a problem. Of course the positive density should also go away in the Fo-Fc map since it is now accounted for by the model. All the best, JP On Friday, July 26, 2024 at 10:03:10 AM CDT, Renuka Kadirvelraj <[email protected]> wrote: Follow up to OP: Presence of negative density in Sim Omit map Hi CCP4bb, Thank you very much for the help and apologies for some missing info from the earlier post, it is here below. Also, thanks to Oleg, John, Edward, Eleanor, Dave and Andrea for their advice, suggestions and things to try. The updated pic (left) shows the misbehaving lysine, the contour level of this Sim Omit map is 3 sigma. The residue is not disordered per se, we had no trouble modelling it; the Sim Omit was to make sure it is a true outlier. We used Wilson scaling while converting the I’s to F’s to approximate the F000. The negative density showed up in the Sim Omit map and only around the loop that was deleted. We did not see a complementary negative density near that loop nor elsewhere in the regular, non-omitted difference map (at 3 sigma or at a lower contour level of 2.7 sigma). We ran a bog-standard difference map like Eleanor suggested; set the loop occupancy to zero and then run cycles of refinement. The positive density for the Lys shows up; unfortunately, the flanking red density shows up as well. Difference density map pic on the right, contour level 3 sigma. Perhaps this is just the complementary sagging around the peak, like Edward suggested with his child-standing-on-water-bed’ analogy. Also, we’re looking into the incorrectly modelled bulk solvent issue that Dave and Andrea mentioned. Thanks again, Renu --------------------------------------------- Renu Kadirvelraj, Ph.D. Research Scientist A428, Biochemistry and Molecular Biology 120, East Green Street University of Georgia Athens, GA 30602 Tel: (706) 583 0303 ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of John Bacik <[email protected]> Sent: Thursday, July 25, 2024 12:21 PM To: [email protected] <[email protected]> Subject: Re: [ccp4bb] Presence of negative density in Sim Omit map You don't often get email from [email protected]. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> [EXTERNAL SENDER - PROCEED CAUTIOUSLY] I also noticed one residue looks like a Phe and there is another that may be a Lys but not a very good view of it. When doing simulated annealing its not outside the realm of possibility that it will introduce errors in other parts of the model that could in theory explain the additional red density. The density does look a bit unusual though and it would be interesting to see if this simply disappears with another round of refinement. I believe you may in certain cases see some red density in for example hydrophobic pockets where in reality there is little or no bulk solvent and it is not properly accounted for in the refinement. JP On Thursday, July 25, 2024 at 01:11:05 AM CDT, Eleanor Dodson <[email protected]> wrote: Hmm - I cant quite understand your map - that density is not for the lysine ? It looks like a well ordered PHE contoured at quite a high level? As Edward suggests - if you omit a well ordered feature the resultant difference maps often show high positive density and a surrounding complementary "sag" of negative density - not really something to worry about.. But I wonder why you don't just do a bog standard difference map? Set the occupancies of the loop you want to omit to 0.00 - do a few cycles of refinement and see what comes back? A prediction - the well ordered features will show up loud and clear and your surface LYS wont! High resolution structures show they often are in multiple conformations, which would be hard to model at 2.1A. And just a thought about negative features in difference maps. I often see random "holes" which don't seem to have any logical explanation.. I sloppily put them down to "data defects - missing data? poorly measured data? etc - but does anyone have a more satisfactory explanation? Or dont other people see them?? Eleanor On Thu, 25 Jul 2024 at 01:48, Edward A. Berry <[email protected]<mailto:[email protected]>> wrote: Does your difference map have mean value ~zero (over 1 ASU or cell)? If maps are constructed by Fourier transform without the 0 0 0 reflection, they have mean of zero (because the mean of a sinusoid over one period is zero). That means that any time you add positive (difference) density, which raises the mean value of the absolute map, the Fourier map has to sink down a little to bring that new mean value to zero. Now if your data were complete except for the 0 0 0 reflection, this would move the floor down everywhere by a small amount to balance the large increase at the local peak, and it would not go below the negative contour limit. But without the ultra-low resolution amplitudes the FT cannot make a constant offset, and instead you get sagging around the peak, like a water-bed sagging around the spot where a child (the peak) is standing. This could lead to the sagging part going below the negative contour and giving you the red density. However I would not have expected the negative density to be quite so localized around the peak - depending on your low-res cutoff. As John Bacik suggested you should check for a mis-modeled part of a symm-related molecule, but if that were the case, you should see the red density even before omitting the loop, and you should see the red density somewhere else on your model. If it just appeared after omitting the loop, it could be due to the map sagging under the weight of the peak, and should go away as soon as the residue is replaced. eab Renuka Kadirvelraj wrote: > Hi CCP4bb, > We would greatly appreciate your advice regarding an odd problem that has > cropped up with one of our > crystal structures. We have a protein structure in space group P6(3)22 at 2.1 > A resolution and in > the final refinement stages with Rwork of 0.20 and Rfree of 0.22. When we ran > simulated annealing to > rebuild an outlier Lysine residue in a loop (using Phenix), the Sim Omit map > showed the positive > density expected from the omission of the loop (colored green in attached > pics). However, flanking > the positive density, there is a complimentary negative density (in red). We > are puzzled as to the > cause of the appearance of the negative density. Can you help us? > Many thanks, > Renu > > --------------------------------------------- > Renu Kadirvelraj, Ph.D. > Research Scientist > A428, Biochemistry and Molecular Biology > 120, East Green Street > University of Georgia > Athens, GA 30602 > Tel: (706) 583 0303 > > ---------------------------------------------------------------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk>, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
