[phenixbb] Problem with R-free assignment

[Jose Arcadio]

Dear phenix.refine community:

I am solving a protein structure using experimental phasing. In order to 
do that I combined two derivative isomorphous datasets into a double SAD 
experiment (to increase the individual weak anomalous power from each 
dataset) using SHARP.My resolution is 3.2 A, and the space group is C2221.

I managed to build a model using the density-modified phases from SHARP. 
Then I started refining against one of my derivative datasets because my 
native dataset is non-isomorphous due to the heavy meal soaking.Then I 
made two mistakes :(

1. I did not realize at the beginning of my refinement-building rounds 
that I was refining against the dataset that showed the worst statistics.

2. I was using a dataset with only FP and SIGFP but not DANO and 
SIGDANO. I used the phases from my SHARP map (also the experimental 
phase restraints)

Now I did a new run of refinement using my best dataset (better 
statistics) and also including the anomalous data FP, SIGFP, DANO and 
SIGDANO. Obviously, my R-work and R-free got pretty much better.

Nevertheless, the R-free was better than R-work at the beginning of the 
run, at the end of the refinement R-free and R-work inverted as usually 
(being R-free worst than R-work)  r_work = 0.2582  r_free = 0.2888 . 
Certainly, there was a mistake with a new assignment of the free set of 
reflections. Now I am aware that the set of free reflections has to be 
the same all over the refining procedure.

Here are the questions

How can I be sure that my R-free is still a safe cross validation of the 
correctness of my structure?

Is it useful to perform a round of simulated annealing in order to erase 
any wrong information in my model and start the refinement rounds again?

Do you have any suggestions?

All the best

Jose Farias
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