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