[phenixbb] Problem with R-free assignment

Jose Arcadio jose.farias at tuebingen.mpg.de
Fri Sep 2 03:00:11 PDT 2011

On 9/1/11 6:49 PM, Pavel Afonine wrote:
> Hi Jose,
> DANO and SIGDANO are not used in refinement.
> Resetting ADPs to an average value, and performing ~5 macro-cycles of 
> refinement with SA and weights optimization typically should be 
> sufficient. Make sure you are using the latest PHENIX version from 
> nightly builds:
> http://www.phenix-online.org/download/nightly_builds.cgi
> Let me know if you have any questions.
> Pavel.
> On 9/1/11 9:25 AM, Jose Arcadio wrote:
>> 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
>> _______________________________________________
>> phenixbb mailing list
>> phenixbb at phenix-online.org
>> http://phenix-online.org/mailman/listinfo/phenixbb

Thanks a for the help Pavel and Nat.

In order to reset ADPs to an average value, shall I use:  set_b_iso=30 ?

Could you suggest a good starting point for wxc_scale and wxu_scale for 
my resolution (3.2 A). Or maybe I should estimate these values empirically?

I would also like to include the heavy metals in my structure, in this 
case I guess it is important to have DANO and SIGDANO as you also suggested.

Best regards


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