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 <
> [log in to unmask]> 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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