Hi Sun
Your bond length & angle RMSD's look suspiciously high for a 2.8 Ang
structure; this usually means that some weighting parameter(s) is/are
not optimal. 2.8 Ang is not that far from the point where the optimal
choice of structure parameters may be torsion angles instead of
Cartesian co-ordinates, in which case the optimal RMSD's for bond
lengths & angles would be exactly zero.
You should be optimising all weights that have been set arbitrarily
by the program (i.e. not obtained from independent experimental
sources), this includes not just the X-ray weight but also the B-factor
restraint weight(s) (the usual culprit) and the NCS restraint weight(s),
as Clemens suggests. I now use the free log(likelihood) to optimise
the weights rather than Rfree, this is now printed by newer versions
of Refmac, but it's up to you which you believe (the difference in the
results may not be significant anyway). Alternatively CNS &
phenix.refine have scripts which automatically optimise the weights
(against Rfree) for you (maybe one day CCP4/Refmac will have this very
useful capability ;-) ).
Also I would check your waters manually - don't believe everything
the auto-water placing software tells you, i.e. does the density
look sensible (at least roughly spherical shaped), is it possible
they are something other than water (check for excess density
and/or suspiciously low B factor), do they all H-bond to protein
and/or other waters you are confident about. I had a structure at
2.9 Ang where I found only 10 good waters and that was for 900 residues
in the a.u. (maybe it had something to do with the fact that the solvent
content and average B were quite high and the data was partially twinned
so the map quality was poor). I'm sure others have opinions on how many
waters you expect to find at various resolutions.
HTH
-- Ian
> -----Original Message-----
> From: Sun Tang [mailto:[log in to unmask]]
> Sent: 04 February 2008 22:32
> To: Ian Tickle
> Cc: [log in to unmask]
> Subject: RE: [ccp4bb] an over refined structure
>
> Hi Ian,
>
> Thank you very much for your detailed information.
>
> I checked the effect of weighter term (wa) in CCP4i for the
> R/Rfree. When I used wa=0.01 , the value is 0.225/0.277
> FOM =0.799. The values changed to 0.204/0.269 (FOM=0.806)
> for wa= 0.05, 0.195/0.268 (FOM=0.807) for wa=0.1 and
> 0.186/0.267 (FOM=0.807) for wa=0.2, respectively. It seemed
> that increase in wa decreases both R and Rfree with R more
> than Rfree.
>
> Which wa value is the best one in this case?
>
> Thank you very much for your valuable help.
>
> Best,
>
> Sun
>
> Ian Tickle <[log in to unmask]> wrote:
>
>
> Hi Sun Tang
>
> Unfortunately there's no such thing as a fixed value
> for the maximum acceptable Rfree-Rwork difference that
> applies in all circumstances, because the 'normal' difference
> depends on a number of factors, mainly the
> observation/parameter ratio, which depends in turn on the
> resolution and the solvent content (a greater solvent content
> means a bigger cell volume which means more reflections for a
> given number of ordered atoms in the a.u. and hence a bigger
> obs/param ratio). The Rfree-Rwork difference also depends on
> Rwork itself (i.e. you tend to get higher values of
> Rfree-Rwork for higher values of Rwork), so it's better to
> think in terms of the Rfree/Rwork ratio (which is independent
> of Rwork).
>
> So for example at very high resolution a 'normal' value
> for Rfree-Rwork might be only 0.02 (so 0.05 which is what
> many people consider acceptable would actually be
> unacceptably high), whereas at low resolution it might be 0.1
> (so 0.05 would be unacceptably low). Also you need to bear in
> mind that Rfree tends to have a quite high uncertainty,
> particularly at low resolution (because it's usually based on
> a relatively small number of observations), so the deviation
> has to be quite big (e.g. > 3 SU) before it can be considered
> to be statistically significant.
>
> So Rfree needs to be compared not with Rwork at all but
> with the value of the optimal Rfree/Rwork expected on the
> basis that the model parameterisation and weighting of X-ray
> terms and restraints are optimal and the errors in the model
> have the same effect as the random experimental errors in the
> data (i.e. a statistical 'null hypothesis'). As Tim just
> pointed out we tried to do this in our Acta D (1998) papers:
> there you can compare your observed Rfree/Rwork ratio either
> with the theoretical value or with the value found for
> 'typical' structures in the PDB at the same resolution.
>
> An abnormal Rfree/Rwork ratio could arise from a number
> of causes, not just over-fitting (I assume that's what you
> mean by 'over-refinement' - it's not clear to me how a
> structure can be 'over-refined' since a fundamental
> requirement of the maximum likelihood method is that the
> structure is always refined to convergence, and refining
> beyond that will by definition produce no further
> statistically significant changes in the parameters).
>
> For example the number of parameters being refined may
> be either too low, or too high (over-fitting), or the values
> of the weighting parameters may not be appropriate, or there
> may be something badly wrong with the atomic model (e.g.
> mistraced chain). Given the values you are reporting I think
> the latter is very unlikely, possibly you just need to tweak
> the X-ray and/or restraint weights.
>
> HTH
>
> Cheers
>
> -- Ian
>
> > -----Original Message-----
> > From: [log in to unmask]
> > [mailto:[log in to unmask]] On Behalf Of Sun Tang
> > Sent: 04 February 2008 16:56
> > To: Boaz Shaanan
> > Cc: [log in to unmask]
> > Subject: Re: [ccp4bb] an over refined structure
> >
> > Hi Boaz,
> >
> > Thank you for your opinions. The resolution is 2.8A and I
> > remembered some people may think the structure is
> > over-refined when the difference between Rfree/Rwork is
> > greater than 6.
> >
> > What do you think the greatest acceptable difference
> between the two?
> >
> > Best,
> >
> > Sun
> >
> > Boaz Shaanan wrote:
> >
> > Hi,
> >
> > Why do you think this structure is over-refined ? The
> > Rfree/Rwork difference of 6.2% seems fine, although you
> > didn't mention resolution. If anything, an over-refined
> > structure would show a smaller difference, as far as I know.
> > If all the other criteria (Ramachandran outliers, etc., map)
> > are OK you should just be happy with your structure.
> >
> > Cheers,
> >
> > Boaz
> >
> > ----- Original Message -----
> > From: Sun Tang
> > Date: Monday, February 4, 2008 18:41
> > Subject: [ccp4bb] an over refined structure
> > To: [log in to unmask]
> >
> > > Hello All,
> > >
> > > I refined a structure with Refmac in CCP4i and the
> R/Rfree is
> > > 0.215/0.277. The difference between R and Rfree is
> > too much even
> > > though I used 0.01 for weighting term in the
> refinement (the
> > > default value is 0.3). The RMSD for bond length and
> > bond angle
> > > is 0.016 A and 1.7 degree.
> > >
> > > What may be wrong with the over-refined structure?
> > What is the
> > > reason for leading to an over-refined structure? How
> > to avoid it?
> > >
> > > Best wishes,
> > >
> > > Sun Tang
> > >
> > >
> > > ---------------------------------
> > > Be a better friend, newshound, and know-it-all with Yahoo!
> > > Mobile. Try it now.
> >
> >
> > Boaz Shaanan, Ph.D.
> > Dept. of Life Sciences
> > Ben-Gurion University of the Negev
> > Beer-Sheva 84105
> > Israel
> > Phone: 972-8-647-2220 ; Fax: 646-1710
> > Skype: boaz.shaanan
> >
> > ΓΆβ'¬Ε½
> >
> >
> > ________________________________
> >
> > Never miss a thing. Make Yahoo your homepage.
> >
> >
>
>
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