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Thank you all for answering with the helpful suggestions I suppose I should explain my situation in a bit more detailed. My 2.1A data has ~55500 reflections with 2800 flagged as Rfree set in CNS. I have not altered the reflection file or switched programs for refinement. The molecule has 2 molecules /AU so I can use NCS restrain on the protein portion only. The structure was previously stalled at Rfree 0.279 and Rfactor ~0.24 and i suspected its overfitted because I had performed bindividual refinement before I had attempted minimize.inp. So I had to start all over again. At that point much of the DNA molecule was fixed quite well so I basically took the coordinates of the fixed DNA molecule and glued it into the pdb file of the structure that was in the very early stage of refinement when it was right after molecule replacement and rigid body refinement step (the protein makes up >70% of the structure). I was able to get my Rfree back to the 0.28 range rather quickly with some manual rebuilding and only short uses of minimize.inp (only 4 cycles each time). I have only 200 water molecules so far and B-factor refinement has not been performed yet. Rfree and Rfactor in CNS are 0.282 and 0.279 respectively. In the next step I had made a DM solvent flipping and NCS averaging map using CNS and basically fitted my protein into the density using graphics program "coot". using especially the real space refine option. I then ran minimize.inp, which show the starting Rfree and Rfactor to be both 0.271. But after 2 steps in the refinement the Rfree increases and Rfactor begins to drop. They finally seperate after 10 cycles to Rfree = 0.276 and R= 0.269. A number of you have suggested that Rfree and Rfactor should seperate a bit after intial rounds refinement and now I see that is making sense. The wa were automatically determined to be 1.78. but my rmsd bonds and angles are 0.037 and 2.45 respectively, which isn't so good. So i guess i did messed up my geometry quite a bit. Perhaps I should go back now and seriously deal with the geometries in the structure. Please shout out any other ideas. Thank you for your responses Jimson On 3/9/06, Jimson Wong <[EMAIL PROTECTED]> wrote: > Hello all > I am trying to do positional refinement on a model of a protein & DNA > complex using minimize.inp from CNS. The problem I am having is that after > 2 cycles of automatic position refinement the Rfree would start to go up. > This happens almost everytime I try to use minimize.inp to do positional > refinement. Through labourous manual rebuilding and with the help of DM > solvent flipping + NCS averaging map the Rfree and Rfactor are both 0.271 at > the moment, with only 200 water molecules along with 680 residues. I could > add more water molecules to drive down the Rfree, but I really would l like > to be able to use minimize.inp to refine the position of the molecules and > so far I cannot do that for some reason. I have made sure that there were > no clashes between non-covalently linked atoms and that their distances are > not closer than 2.2A. > > Would any experienced CNS users know what it means when the Rfree starts to > go up when one uses minimize.inp? > > Jimson > >
