Hi George, the target function implemented for real-space refinement is such that it moves atoms towards closest map peaks (the target is the negative sum of map values calculated at atomic centers). Obviously, this target is not very similar to R-factor function or reciprocal space targets that are more or less quadratic near the minimum. All in all this means that real-space refinement does not guarantee R-factor decrease and a following round of reciprocal space refinement is necessary if you plan to look at R-factors. The reason why this target is use is because it is fastest to calculate. Pavel On 9/19/14 1:22 PM, Nathaniel Echols wrote:

Global real-space refinement simply isn't very useful at high resolution, and you shouldn't need a large radius of convergence when the R-free is already 13%. I'm a big fan of automating everything possible, but this is one case where fixing the sidechains manually in Coot (which should do an excellent job) is by far the easiest option. Once you fix the sidechains the C-beta outliers will probably disappear.

PS. A common reason for C-beta deviations at high resolution is residues with alternate conformations being split at C-beta rather than splitting the entire residue, which allows the backbone to move to accommodate the optimal placement of the sidechain (rather than straining the geometry to get the sidechain atoms to fit the density). Always split the entire residue - the backbone is almost never rigid in disordered regions.

On Fri, Sep 19, 2014 at 1:11 PM, George Devaniranjan mailto:[email protected]> wrote:

Hi,

I refined (phenix.refine) a 1.0Å structure but it had a few CB outliers and some rotamer outliers.

I decided to try real_space_refine, to check if they could be fixed (given that it has a higher radius of convergence). (using run=local_grid_search+minimization_global in real_space_refine)

But, the R/R(free) increased from 11.39/13.24( for the refine model) to 16.51/17.55 (real_space_refine model).

It seemed a big increase, when I looked at the map against the structure the protein itself seemed fine. The rotamers outliers had decreased, the CB outliers were fixed..etc

The solvent fit to the density had worsened in COOT.

What am I doing wrong? Any suggestions, advice would be much appreciated.

Thank you, George

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