Hi Derek,

choosing 5% for free set is not a dogma. I always use 10% and that's what CNS was doing for years. In your case this will make 200. Not a whole lot but better than 100.

You can generate several (say 10-50) different test sets and independently refine the model against each of them (from the very beginning). Then make a note of differences (in model, R-factors). Those differences will be uncertainties likely due to different test sets used.

I realize it may be tedious to do 10-50 refinements per each model parametrization and refinement strategy that you want to test. In this case I would simply reduce choices down to most reasonable given the resolution and model quality:

- use individual B-factor refinement. With type of restraints we have it is ok to do in most cases. Switch to group B refinement only if you have strong reasons to believe that individual B refinement isn't good for your case.
- Use torsion NCS;
- Use Ramachandran plot restraints only to keep (preserve) good conformations during refinement, not to fix bad ones (outliers). That is: in case of outlier, for it manually first then refine with Ramachandran restraints so that it does not become outlier again.
- If you have a higher resolution good model, you can use it as a reference model, if needed.

In future we will investigate using ideas recently published in Acta D that suggest ways to overcome the problem of too small test sets.

Pavel

On 12/19/14 3:18 AM, Derek Logan wrote:

Hi everyone,

Right now we have one of those very difficult Rfree situations where it's impossible to generate a single meaningful Rfree set. Since we're in a bit of a hurry with this structure it would be good if someone could point me in the right direction. We have crystals with 1542 non-H atoms in the asymmetric unit that diffract to only 3.6 Å in P65, which gives us a whopping 2300 reflections in total. 5% of this is only about 100 reflections. Luckily the protein is only a single point mutation of a wild type that has been solved to much better resolution, so we know what it should look like and I simply want to investigate the effect of different levels of conservatism in the refinement, e.g. NCS in xyz and B, group B-factors, reference model, Ramachandran restraints etc. However since the quality criterion for this is Rfree I'm not able to do this.

I believe the correct approach is k-fold statistical cross-validation, but can someone remind me of the correct way to do this? I've done a bit of Googling without finding anything very helpful.

Thanks

Derek

________________________________________________________________________
Derek Logan                                         tel: +46 46 222 1443
Associate Professor                                 mob: +46 76 8585 707
Dept. of Biochemistry and Structural Biology              www.cmps.lu.se
Centre for Molecular Protein Science            www.maxlab.lu.se/crystal
Lund University, Box 124, 221 00 Lund, Sweden           www.saromics.com

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