Dear Colleagues, I'd like to share with you with a twinning puzzle that's been troubling me for a while. I have a ~2.2 A data merged in p212121 space group with unit cell: 41.8300 117.4490 134.4840 90.0000 90.0000 90.0000 Translational NCS was detected but structure solution was able to be obtained by phased molecular replacement as we have a Au derivative data set. Obtaining experimental phase or MR alone didn't work. However, with this model and data set, the refinement didn't work (with R-free ~40% or higher if with more cycles). Different twinning tests have been carried out and no sign of twinning. All possible p222 space groups have been tried but no luck or even worse. Considering that translational symmetry and perfect twinning may make things deceiving, the data was merged into p21 and a twin-refinement was performed in phenix.refine. After 3 cycles of rigid-body and group ADP, a R-free of ~30% was obtained and the output map looks OK. It looks like we got it. But then things get interesting when we tested other two axis as the p21 unique axis. With all the three choices plus corresponding twin-refinement, we could get a similar R-free (30%). Besides, the three maps all look OK except for one missing some side chains. Then the structure was solve in p1 and Xtriage was used with the hope that one axis would stand out. However, the RvsR for all three axis are almost the same. Did I miss anything here? Of course, we could pick up the one axis we like best and get the structure solved ^---^. However, this puzzle would eat me alive for a very long time. Best, Hua