Refinement
Theory
For now, see http://www.usm.maine.edu/~rhodes/ModQual/index.html#RefineXray
Programs
- Refmac
- CNS
- phenix.refine
what can go wrong in refinement?
R-factor does not go down
If this happens in the R-factor range of 30-40, here are a couple of possible reasons:
- wrong spacegroup (usually the true symmetry is lower; see below)
- twinning (this happens more often than you'd like, see A. A. Lebedev, A. A. Vagin and G. N. Murshudov (2006) Intensity statistics in twinned crystals with examples from the PDB. Acta Cryst. D62, 83-95)
- bad data - check the statistics of the data reduction program
- model incomplete or wrong
- other refinement options should be exploited, e.g. try TLS refinement
help, my protein has high B-factors!
This is also a FAQ on CCP4BB. The answer is: there's probably nothing wrong with it. If your crystals diffract to 3 A at a synchrotron, then the average B-factor should most likely be on the order of 100 A^2. If your crystals diffract to 2 A, then the average B-factor is most likely on the order of 40 A^2 or so. Use B. Rupp's calculator ([1]) to find out the dependance of scattering power on B-factor.
R_free much higher than R
how large should the difference between R_free and R be?
Wrong space group
Sometimes crystal symmetry combines with non-crystallographic symmetry (NCS) and produces a diffraction pattern resembling higher symmetry space group than what you really have. NCS in this case closely resembles crystallographic symmetry. If resolution is not high enough, the difference in spot positions may be too small to give any detectable problems with indexing, integration and scaling. Even phasing (e.g. molecular replacement) may be successful. But if your R-factor hangs fairly high and you have problems building parts of your structure, it is worth trying to check other space groups. The most straightforward approach is to try processing data in P1, because if that does not bring R-factor down significantly, other space group choices will not solve the problem either.
This occurs most often at moderate resolution. However, the structure of the ketosteroid isomerase had to be refined in P1 at atomic resolution, although it refines well in C2221 at lower resolution such as 1.5A.