Dear Jim,

One thing that you did not mention was the resolution of the structure that you used as a model for MR. We have had numerous cases of solving a structure by MR where the Rfree has been lower than one might normally expect for the resolution we were working at in cases where our MR model had been refined at substantially higher resolution than the new structure. For example, when the MR model had been refined at 2Å, but the structure we were solving only had data to 3.0Å. 

I rationalise this by the fact that the starting model was actually better than one would normally expect to get working at 3.0Å resolution. Fortunately I have never had a referee saying that the Rfree was too low !

Another point is that the Rfree can also be lower than expected if there is a very high solvent content. This is because the high solvent content results in significant correlations between structure factors of different reflections and this means that the Rfree set is no longer completely "free", in an analogous way to having high NCS present.

I agree with others that you need to go back to the editor about this, the referee's condition seems completely unreasonable.

Best wishes,

Andrew

On 26 Apr 2016, at 21:32, Professor James Henderson Naismith <[log in to unmask]> wrote:

Dear Colleagues.

We are having difficulty persuading a reviewer that our structure is not over refined.

The structure is a molecular replacement of complex with a published relatively non-isomorphous native structure from another lab.

The same Rfree set was used as the published data.

Our complex is at 2.6A and R/Rfree end up at 18/22

PDB redo gets the same result, so does phenix.refine (with a trivial %). All B-factors were reset and TLS used.

The data are 2.61A and average B is 80A, there are 4500 residues, 68 waters. Unfortunately Mol probity gives us 100th centile and the Rama is also good, bond rms is 0.012 and we used NCS local restraints.

There is no rotational NCS but there is a weak translation symmetry (does not show up in data but when refined the monomers have a bead on a curved string appearance).

The referee has refused accept the paper until we make the R and Rfree higher by some undefined target, since it is 'over refined'

Does anyone have a useful program to make structures worse to some threshold that is considered normal at 2.6 A or does anyone know a good paper that points out Rfree is not susceptible to over refinement since by definition it is not refined.

best

Jim

Jim Naismith

St Andrews

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