Hi all Thanks for all your reply, really helpful Joe wrote,

My first guess is that the R-free set generated in CCP4 is not set to follow twin laws, so you have work and free reflections associated by twinning. However, I would expect PHENIX >to check this.

As for the reduced size, that probably comes from the way the twinned test set is calculated. Maybe it is a bug. Are you using anomalous data?

No anomalous

What is the PHENIX version?

Peter wrote,

- you have at least n% of free flags, but not more then 2000+epsilon

(typically)

- it follows any possible twin law (not user specified, it follows symmetry of the lattice)

After I change the setting "xray_data.r_free_flags.max_free ", and set it up to 5000. xray_data.r_free_flags.fraction=0.05, the results look normal. Final: r_work = 0.1782 r_free = 0.2285 bonds = 0.007 angles = 1.186 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 R VALUE (WORKING + TEST SET) : 0.1807 REMARK 3 R VALUE (WORKING SET) : 0.1782 REMARK 3 FREE R VALUE : 0.2285 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.07 REMARK 3 FREE R VALUE TEST SET COUNT : 2776 Best Regards Lauren