If you are doing real-space refinement of the entire model against a map in coot, isn't it also important to make sure that the Rfree reflections were NOT used in calculating that map? Depending on how the map was made, that info can be hidden in the fine print.
++++++++++++++++++++++++++++++++++++++++++
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; [log in to unmask]
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp
________________________________________
From: CCP4 bulletin board [[log in to unmask]] on behalf of Robbie Joosten [[log in to unmask]]
Sent: Monday, April 21, 2014 4:40 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] coot problems to decrease R FREE
Dear Peter,
> I'm a novice of coot and ccp4. Now I'm doing refinement using both refmac5
> and coot.Here are some problems I'm facing. Really hope you can give me
> some suggestions.
>
> 1、THE RESOLUTION OF THE DATA IS 2.5 angstrom. After first refinement of
> refmac5 I got R factor which is 0.26 and R FREE which is 0.31. My question
is
> what the final R factor and R FREE should be after several rounds of
> refinement by refmac5 and coot.
As low as it can reasonably be. Which is of course a lousy target but the
purpose of model refinement is to make it as good as possible, not to
achieve a certain R-free. That said, at this resolution, the PDB average is
about 25% for R-free.
> 2、At which map level（e/A3 or rmsd）should I refine the data by coot?
You refine against the whole map, but for viewing purposes you should change
the map contour level during building. For the majority of the map you
should be able to see stuff well above an rmsd of 1.0 for the 2mFo-DFc map.
At the difficult bits you may need to go below 1.0, but no so low that you
can fool yourself. The proper height for the difference map depends as a bit
on the situation. I usually just turn it down until obvious noise peaks
(small negative peaks in the solvent) appear. That typically ends up
somewhere between 3 and 4 rmsd.
> 3、Can you give me some tips and strategies about how to use coot to
> decrease R free? now I just use some basic tricks such as fit density and
> Ramachandran plot to refine the data.
This is what I do for your kind of resolution: set the weight for refinement
in COOT to 50 or 40 (the default is 60) and switch on torsion restraints and
possibly Ramachandran restraints. Then I go through the entire structure
residue by residue (space bar) refining windows of three residues (with the
't' key) and fixing everything that I can (that includes obvious solvent
molecules). Symmetry should always be switched on and NCS ghosts (if
available too). When that is done you can focus on the remaining difficult
bits by looking at the difference map peaks in COOT and at validation
reports from WHAT_CHECK, and MolProbity.
So now for the bit where I plug my own stuff: you can try PDB_REDO
(http://xtal.nki.nl/PDB_REDO for the server, there is also a stand-alone
version) to take a lot of work out of your hands. It optimises your
refinement in Refmac and, rebuilds side-chain and tries to find peptide
flips that improve you Ramachandran plot and fit with the maps. You also get
a lot of validation information that may help you with further rebuilding of
your model.
Cheers,
Robbie
>
>
> Best regards,
>
>
> Peter Chen
>
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