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
<0000de831e80596f-dmarc-requ...@jiscmail.ac.uk> wrote:
#yiv4749023127 P {margin-top:0;margin-bottom:0;}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
ScientistA428, Biochemistry and Molecular Biology120, East Green
StreetUniversity of GeorgiaAthens, GA 30602Tel: (706) 583 0303From: CCP4
bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of John Bacik
<0000b45abf420e1f-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, July 25, 2024 12:21 PM
To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Presence of negative density in Sim Omit map
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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.
JPOn Thursday, July 25, 2024 at 01:11:05 AM CDT, Eleanor Dodson
<0000176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> 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 <eaber...@gmail.com> 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
>
> ----------------------------------------------------------------------------------------------------
>
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