Dear All, I am stuck with this problem for 2 months and hope you could help.
We have a 2.1 A dataset for a 380 amino acid long protein. The space group is I4 (single molecule in asymmetric unit, 48% solvent content) and the dataset is quite perfect (no obvious pathologies). The protein itself is organised in 2 lobes (N and C terminal lobes). The sequence identity to nearest homologue structure is 17%. We could get the phases by SeMet SAD phasing (3A resolution dataset, 5 SeMet (excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe and 2 partial occupancy (~0.5 each; present on surface) SeMet in N-terminal lobe). Automated model building (at 2.1 A) yielded nice model for the C-terminal lobe (215 residues) and manually I could build parts (around 80 residues) of N-terminal lobe with high confidence. In addition we could also build a ligand which is sandwiched between C and N terminal lobe. However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some patchy density left at the N terminal lobe but as it is discontinuous, I cannot build anything in it (except lots of water molecules). In total I am missing around 85 residues. These residues are predicted to be present in secondary structure (and not flexible). As I have around 75-80% model built, I would expect that I would have all the phases and should get nice density for the remaining part. But as I dont see it, could the rest part be flexible? But again, this is not reflected in the R factors (I would then expect low Rfree). Could it be that I still lack phases (due to partial occupancy of SeMeth in N-terminal lobe ) and have to try to get them by heavy metal soaking, or there is disorder in the N-terminal lobe? I have also tried solving different datasets for same crystal but this has not been useful. Regards, Pravin.
