I have a structure refined to 2.5 A.  After simulated annealing (start temp = 5000, cool rate = 25, final temp = 100, include TLS refinement and NCS restraint), my R and Rfree are 24.6% and 28.7% respectively.  I then turned off the SA in the generated def file and carried on the combined refinement.  My R and Rfree went up to 25% and 29.2% respectively.  Why did the Rfree go up after refinement?

In the remarks of the output pdb file, I have one line, "REMARK    twinned    :  True".  What does this mean?  

The RMSD of bonds and angles after either SA or combined refinement are about 0.006 and 0.851 respectively.  Don't you guys think the geometry restraints are too tight?  How do I change the weight in PHENIX?

Thanks.

Jianghai