Hi,

On Fri, Feb 06, 2009 at 08:13:29AM -0800, Andy Millston wrote:
> Restrained refinement does a good job at refining most of the
> structure except a small region with poor density. Every time I
> refine it, it puts this region completely out of the map.

Do you mean:

  a) after restrained refinement the 2Fo-Fc map tells me the model
     should be were it was before refinement (and were it stays during
     rigid body refinement)

or

  b) after restrained refinement the 2Fo-Fc map and the model don't
     match up.

If it's a), i.e. 2Fo-Fc map before and after restrained refinement is very
similar but model has been moved away: are you sure there isn't some
very bad contact that pushes your model away? Have you checked e.g in
Coot with all symmetry-equivalents switched on?

If it's b): maybe the initial 2fo-Fc map (after rigid-body refinement)
was heavily biased and showed you an artefact? And the restrained
refinement is actually telling you the truth - your model isn't were
you thought it should be and there is no data/information to put it
there.

> It doesn't
> have much effect on overall geometrical factors as its a very small
> part of a relatively large molecule.
> I am annoyed at the fact that Refmac doesn't seem to have much
> respect for the electron density map while refining this region,

But it does (well: it doesn't look at the density per se - for that
you would need to do real-space refinement): just that something else
(probably one of its geometry terms) it telling REFMAC to do what it
does in the end.

> and all my attempts to keep the residues within the map go in vain.

Remember that the map isn't something fixed (unless you have very good
experimental phases): it's based on the model phases after all.

> The refined residues are still ok according to Fo-Fc map, but
> they're completely out of the 2Fo-Fc map boundaries.

What do you mean with '2Fo-Fc map boundaries'?

> Also, my model and the refined model both look bad on Ramachandran
> plot.

That's ok, we're not refining against Ramchandran plot (or at least we
shouldn't) - so as others have mentioned: this is telling you
something.

> But I think its a unique feature of the molecule, at least thats
> what the density suggests. Also, packing score and quality value
> plot suggest that there is nothing seriously wrong with my model.

A lot of these criteria might not pick up details in small
regions. Furthermore, if you start e.g. from a good initial model
(molecular replacement) then all the purely geometrical quality
indicators will be great, since nothing has changed there after
rigid-body refinement.

> As you've suggested I should try altering the restraints and see how it goes.

Or combine restrained refinement for most of your protein with this
part as a rigid body? I don't know if REFMAC can do that though ...

Or leave that part out for the moment and concentrate on getting
everything else as good as possible: this would give you the best
phases frmo the 'good region' and maybe then the 2Fo-Fc and Fo-Fc map
tell you what's going on in the other part. Sometimes its very
beneficial to remove unclear regions again to not bias your own
expectations of what it should look like.

Cheers

Clemens

-- 

***************************************************************
* Clemens Vonrhein, Ph.D.     vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--------------------------------------------------------------
* BUSTER Development Group      (http://www.globalphasing.com)
***************************************************************