[phenixbb] Does PHENIX exclude Rfree reflections in maps?

Pavel Afonine PAfonine at lbl.gov
Wed Sep 30 18:09:01 PDT 2009

Hi Joe,

> Normally, 5% for R-free is sufficient. 

did anyone studied this and came to this conclusion (publication?)?  I'm 
not aware.

In fact, the absolute number is important. The number of test 
reflections per relatively thin resolution shell has to be not smaller 
than 50. This assures that the determination of maximum-likelihood 
target parameters (alpha/beta, or sigmaa) is well defined. More - 
better, but too much is not good since excluding too many reflections is 
not good too. If interpolation is used, then less than 50 can be used (I 
guess what CNS is using), but I have reasons to not like it.

When phenix.refine creates test reflections, by default it is 10%, but 
not more than 2000.

> Even though you may not do 
> real-space refinement with free reflections, external tools can do that 
> with the maps written out. 

It is the most efficient to combine local and global real-space 
refinement with reciprocal space refinement. I call it dual-space 
refinement. This is why it is tightly integrated into phenix.refine:


> I am sure that many people will use it when 
> they find that real-space fitting and refinement tools lower R-free, 
> unaware that they are cheating.

Sure. This is why free-R flags are not used in maps calculation for 
real-space refinement (my previous email).

> With 10% test reflection, I suspect that difference maps used to find 
> waters can easily find a few noise peaks with significant R-free 
> contributions. 

- I'm not aware of any systematic study on this matter, although I can 
believe it in theory;
- phenix.refine uses very sophisticated filtering tools;
- I guess at some point I will switch to using Average Kick Maps for 
water picking. This will remove the noise peaks, and so eliminate the 
problem (I need to test this all, though).

> IMHO, using test reflections for anything but computing R-free should 
> always be avoided unless you are unable to proceed using only the 
> non-test. Using test reflections is always cheating to some extent, 
> although trivial amounts of bias are probably removed during refinement. 
> I just think it is better to be very strict about test reflections and 
> avoid the possibility of bias.

Test reflections are used for calculation of m and D in 2mFo-DFc and 
mFo-DFc maps, as well as in alpha/beta parameters of ML target. This is 

> Exclusion of test reflections ought to be an option. Ideally, deposited 
> PDB files should report whether maps used for model building included 
> test reflections.

Ideally yes, but I can name a hundred of other similarly important 
parameters to report. *At least a set of all parameters must be reported 
so the published R-factors are 100% reproducible - I'm sure this is an 
easy doable goal to start with.*


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