Hi Everyone,
I collected two datasets of the same ligand bound structure at 2.4 and 2.1 Å resolution. The apo structure has already been solved to 2.2Å resolution in P31 (and P3121 for SeMet). After processing each dataset as both P31 and P3121 in MOSFLM, I proceeded with MR with Phaser using sequential partial search models to place either 3 copies in the ASU for P3121, or 6 copies for P31. (The previously solved apo structure had 6 copies of the same protein in the ASU).
*** in summary two datasets, processed as both P31 or P3121
After running xtraige for either .mtz, I get this warning about NCS:
Largest Patterson peak with length larger than 15 Angstrom:
Frac. coord. : 0.333 -0.333 -0.153
Distance to origin : 56.728
Height relative to origin : 26.388 %
p_value(height) : 2.659e-03symmetry.
Translational pseudo-symmetry is very likely present in these data. Be aware that this will change the intensity statistics and may impact subsequent analyses, and in practice may lead to higher R-factors in refinement.
Regardless, I can proceed with the MR using sequential partial search models to place either 3 copies (P3121) or 6 copies (P31) with decent solutions (i.e. packing looks good upon inspection, scoring looks appropriate -> SOLU SET RFZ=2.8 TFZ=8.3 PAK=13LLG=-37 LLG=2199 as an example). However, I did get the following somewhat expected warnings:
Number of known components (1) not divisible by 2: Translational NCS correction not applied
Large non-origin Patterson peak indicates that translational NCS is present
Upon any attempt at initial refinement (with a number of different strategies), the R-factors are hung up > 0.42 (for example in the P3121, 2.1Å case):
Start R-work = 0.4966, R-free = 0.5133
Final R-work = 0.4231, R-free = 0.4477
*** best R-factors from ~ 15 different strategies
My concern is that there is some pathology that is not being recognized (merohedral twinning) or that the NCS/pseudosymmetry is not being addressed appropriately. Any suggestions in troubleshooting this would be much appreciated, as I have pretty much exhausted everything I could think of.