[phenixbb] phenix real space refine & free R reflections
Edward A. Berry
BerryE at upstate.edu
Fri May 5 07:54:41 PDT 2017
On 05/05/2017 05:01 AM, Hillen, Hauke wrote:
> Dear Phenix bb,
> I have a rather conceptual question regarding the treatment of free R flags in phenix.refine and phenix.real_space_refine. I am refining an xray structure using phenix.refine (but not using the real space refinement implemented there) and at some point decided to play a bit with the standalone program phenix.real_space_refine. In particular, I was interested in testing real space refinement against different maps (experimental map vs. model-phased). Being used to the excellent handling of free R flags in Phenix, I assumed giving phenix.real_space_refine the output MTZ of a previous phenix.refine run as a map would be safe to do. But I then saw the phenix.refine FAQ states the following:
> "Are R-free flagged reflections included in the maps used for real-space refinement?
> No, this is almost guaranteed to bias R-free; these reflections are removed internally prior to map calculation. However, the output maps will include these reflections unless you explicitly request otherwise.”
> While it makes sense to me that including the free reflections in the maps used for real space refinement will bias R-free, I wonder why the default behavior of phenix.refine is to output maps which include these reflections. I assume most users will use these output maps (for example via the OPEN IN COOT button in the GUI) to iteratively improve their model by local real space refinement in Coot. Conceptually, how is real space refinement performed by phenix.refine (where the free R reflections are not used to calculate the map to refine against) different from manual local real space refinement in Coot? Will the latter not also bias R-free when the default output maps from phenix.refine are used?
I would argue just the opposite- free reflections should be included in the maps used for RS refinement. Excluding free reflections is the same as setting them to zero amplitude, so you are still biasing these reflections, just biasing them toward a drastically wrong value (zero) instead of biasing them toward fobs.
If the evidence for bias is that R-free gets "better" when you include the free reflections, isn't it more because you avoid biasing them toward zero than because you do bias them toward the Fobs? In the case of density modification, which has the same problem, Ivan Rayment* showed the importance of using the "fill-in" method for missing reflections in making maps. That would seem to be the correct way of implementing R-free for density modification or real-space refinement: you don't give away your Fobs, but at the same time you don't keep forcing the free reflections toward zero.
*Acta Crystallogr B. 1983 Aug;39(4):505-516.
Acta Cryst. (1983). A39, 102-116
More information about the phenixbb