On Sun, Jul 8, 2012 at 2:11 PM, James Garnett <[email protected]> wrote: > I have found a molecular replacement solution in I212121 using an NMR > structure of the same protein and MR-ROSETTA/PHENIX (PHASER LLG=128 > TFZ=12.3), although I can not refine this below R ~45% and Rfree ~50%. The > maps look OK in parts but in other regions the connectivity is much reduced. > In case of model bias I have used density modification and also used > simulated annealing etc in case it is stuck in a local minima - these did not > help. This protein is an Ig-like fold (potential for pseudo-internal > symmetry) and so I have also played around with rotations of the structure > but this has not helped. Although twinning analysis in all spacegroups > suggest there is no twinning I have tried refinement in PHENIX and REFMAC > using twin laws but this does not help.
Several questions: 1) Are you certain you crystallized the protein you're interested in and not a contaminant? This is unlikely to be the culprit, but it's always good to check. (An R-free of 50% does not guarantee that the model is correct.) 2) What happens if you delete the regions of the model where the map connectivity is poor, and refine the partial model? 3) Did you try rebuilding the model completely from scratch, i.e. starting from the map without an input PDB file? I'm pretty sure MR-Rosetta will only do this if the sequence is significantly different than the template. I'd recommend trying several different programs to do this (AutoBuild, ARP/wARP, Buccaneer), as the methods involved are quite different, and you may be able to combine different fragments together afterwards. -Nat
