Dear Thierry,
This is a very interesting approach and observation. Using a
threshold of 0.6 rmsd, if converted into a ratio of B-factors, would
mean accepting waters that have a 40% higher B-factor than would be
done with a 1.0 rmsd threshold.
Philippe Dumas commented off-list on my anecdote regarding the
extra copy of a molecule only becoming convincingly visible at 0.35
rmsd, and he pointed out that it could have been a case of partial
occupancy rather than of a uniformly higher B-factor. That is a very
good point, which we haven't pursued. However the conclusion is the
same: the contouring level at which a density map is examined must be
adapted to the model of disorder for that region that one is prepared
to consider: for an increased B-factor, using the formula I outlined;
or for a partial occupancy, using a corresponding fraction of the
contour level at which full-occupancy regions are examined. The main
thing is that the contour level in rmsd units isn't the same thing as
a signal-to-noise ratio.
With best wishes,
Gerard.
--
On Wed, May 27, 2015 at 01:01:50PM -0400, Fischmann, Thierry wrote:
> Here is another observation regarding the subject of which rms value is more
> sensible to review a structure.
>
> The purpose of the calculation described below was to determine the optimum
> e- density value for rejecting waters.
>
> The principle of the test was very simple : waters were added using the
> "traditional" peakmax / H-bond distance based criteria. The cut-off in
> difference peaks for water selection was deliberately set to be "low" (I
> forgot what it was, as it's been a while since I've performed these tests).
> The structures were refined, then waters were rejected if the sigmaa-weighted
> 2Fobs-2Fcalc density value was less than a specific threshold. A contact
> check was also used for water rejections. The structure was then given
> another final round of positional refinement. All refinement were performed
> using BUSTER. The Rfree values were then compared : the optimum e- density
> cut-off value would be - obviously - the one for which the value of Rfree is
> lowest.
>
> The tests were performed with a few datasets, all good resolutions although I
> can't remember what they were (definitely better than 2Å). One caveat was
> that these tests were performed only for one project i.e. human Cdk2 kinase.
> A consistent set of "free" reflections between datasets and the one from the
> PDB used as starting point was used, as you would expect.
>
> The best cut-off according to the Rfree criterial was consistently about 0.6
> rmsd. It most likely could vary depending on the crystal system and the
> resolution. But in the simple scenario described above the "best" value was
> quite a bit lower than the "traditional" cut-off of 1.0
>
>
> Thierry
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Gerard
> Bricogne
> Sent: Wednesday, May 27, 2015 8:32 AM
> To: [email protected]
> Subject: Re: [ccp4bb] Validity of Modeling of Highly flexible region at low
> sigma levels
>
> Dear Sagar and Pavel,
>
> Yes, it may be model bias, but you also have to bear in mind that
> the rmsd that is used as a unit in the choice of contouring level
> should not be thought of as the standard deviation of a Gaussian
> random variable and therefore implicitly associated to a "significance
> level" relative to a noise level. This is unfortunately a widespread
> misconception.
>
> We have seen a case where an entire copy of a molecule had been
> missed at first because its density was visible only at a contour
> level of 0.35 "sigma", which at first sight would make anybody shrink
> back in horror. When it was nevertheless modelled and refined, there
> was no doubt whatsoever that it was there, making perfectly sensible
> contacts with its nearest neighbour molecules, obeying good NCS with
> the already placed copies and markedly improving the fit to the data.
> The peculiarity of that extra copy was that a clash around a symmetry
> element with its symmetry mate caused it to have a mean B-factor that
> was twice that of the other, well-ordered, copies of the molecule.
>
> Now, if you look at the expression for the Debye-Waller factor in
> real space, you will find the B-factor raised to the power 3/2 in the
> denominator. That means that the blurring effect of that D-W factor in
> real space results in a lowering of the maximum value of an atomic
> electron density to which it is applied by B^(3/2).
>
> The consequence is that electron density for a molecule whose
> mean B-factor is double that of another molecule will look similar at
> a contour level of 1.0/(2^(3/2)) = 0.35 to what the other molecule
> looks like when examined at a contour level of 1.0.
>
> It is therefore a good idea to remember that the "sigma" level is
> mainly determined by the better-ordered region(s) of the molecule(s)
> present in the asymmetric units, and that less-well ordered regions
> will automatically have a contouring level handicap that is not to be
> automatically interpreted as a lower level of significance. Omit maps
> are a good idea of course, but do bear in mind the (B/B0)^(-3/2)
> effect, where B0 is the mean B in the well-ordered region, and B is
> the same quantity in a less well ordered one. Above all, don't confuse
> "sigma" with a noise level!
>
>
> With best wishes,
>
> Gerard.
>
> --
> On Wed, May 27, 2015 at 12:18:25PM +0100, Pavel Afonine wrote:
> > Sagar,
> >
> > what you see may be model bias unless you calculated these maps without
> > that region of molecule.
> >
> > Pavel
> >
> > On Wed, May 27, 2015 at 12:03 PM, Sagar De'Biomimic <
> > [email protected]> wrote:
> >
> > > Dear all,
> > >
> > > We have solved a structure of Protein-DNA complex. In an ASU we have two
> > > protein homodimers and two DNA duplexes. Additionally we were able to
> > > model
> > > N-terminal region (NTR) missing in the previously reported structure.
> > >
> > > I had modelled poly-alanine chains in the positive density and refined.
> > > These chains were then joined to form NTR. Finally the entire NTR was
> > > refined for occupancies.
> > >
> > > I am doubtful about the validity of modelling of NTR as i had dropped down
> > > sigma levels to as low as 0.8 (as i could not see much of the noise) of
> > > FO-FC and modelled in positive density. After refinment the region shows
> > > the density at 0.5 sigma level of 2FO-FC. I have attached a pdf file
> > > showing snapshots of NTR at 0.4, 0.5, 0.6, 0.7 sigma level of 2FO-FC map.
> > >
> > > I would like to know crystallography community's opinion on validity of
> > > such modelling.
> > >
> > > This NTR region is highly flexible. Main intention of modelling it in such
> > > a weak signal was to complete the model for our Molecular dynamics
> > > studies.
> > >
> > > Additionally we have confirmed with SAXS DATA using Ensemble optimised
> > > modelling (EOM 2.0), the high flexibility and multiple conformations of
> > > this NTR region.
> > >
> > > I would like to know if there is a possibility of having a tool similar to
> > > EOM to model highly flexible regions of a protein in electron Density. As
> > > far as I know xMDFF does better at lower resolutions but fails to model
> > > highly flexible regions such as ours.
> > >
> > > Thank you.
> > >
> > > Regards
> > >
> > > Sagar
> > >
> > >
> > >
> > > --
> > > Sagar Khavnekar
> > > Project student,
> > > Structural Biology and Molecular Biophysics lab,
> > > UM-DAE Centre for Excellence in Basic Sciences
> > > University of Mumbai, Vidyanagari Campus, Kalina, Santacruz (East)
> > > Mumbai 400098, India.
> > >
>
> --
>
> ===============================================================
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> * Gerard Bricogne [email protected] *
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