If you have nearly C3 ncs symmetry parallel to the crystallographic 3-fold, you very likely also have some form of pseudo-translational symmetry. If present this will lead to quite a few of your intensities being very small, and poorly measured. This situation can require you to continue restraining ncs even at 2.1A resolution, particularly if many of your weak reflections are "unobserved". It is quite possible that the free R is telling you exactly what you need to know, that your structure factors alone are insufficient to define your model. Dr. Echols' comment:
It may be possible that the NCS restraints are biasing your test set, leading to the suspiciously low Rfree-Rwork gap, but I thought this only applied when the NCS approached crystallographic symmetry, and
is the reverse of my understanding. The cross-talk between the working set and test set is minimal when the ncs approaches crystallographic symmetry, and at its worst when the two are unrelated. When the ncs and "cs" are similar the ncs images of a test set reflections falls very near its proper symmetry images, which are also in the test set by definition. When the two types of symmetry are unrelated, the ncs image of a test set reflection will fall near a reflection which only has a 5% chance of being in the test set. Now you have a direct relationship between a reflection in the test set and one in the working set. With care, a test set can be selected that minimizes this crosstalk. Since you have ncs symmetry that is masked by cs symmetry this is not much of a problem for you. (I don't quite understand how the 2 copy ncs of the DNA relates to the crystallographic symmetry, so it might be a problem.) Dale Tronrud Maia Cherney wrote:
Thank you Nat, Pavel,
Actually, the NCS approaches a 3-fold crystallographic symmetry axis with 3 types of protein molecules plus 2 restraint groups for DNA molecules.
The result is the same with "optimize_wxc=true optimize_wxu=true" options, Rfree is going up without NCS by ~1.5%. Rwork is practically the same.
Should I increase the test set (now it's 5%)? or should I leave NCS=true for the final pdb?
Maia
Nathaniel Echols wrote:
On May 4, 2009, at 6:48 PM, Maia Cherney wrote:
The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints.
This isn't necessarily true - regardless of which program you use to refine, R-work isn't actually the refinement target, and minimization algorithms aren't foolproof either. When you remove the restraints you are effectively decreasing the observation:parameter ratio, which increases the risk of overfitting. If nothing else I would expect the gap between Rwork and Rfree to increase, but I've also seen both increase when NCS restraints were removed.
It may be possible that the NCS restraints are biasing your test set, leading to the suspiciously low Rfree-Rwork gap, but I thought this only applied when the NCS approached crystallographic symmetry, and there's no such thing as fivefold crystallographic symmetry. Hopefully someone else on the list can clarify this.
-Nat
------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]
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