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I disagree with Herman that 3 sigma features are "nothing to worry about". Indeed, I think this is one of the deepest and most disturbing problems with macromolecular crystallography in general. Why can't we explain our data to within experimental error? If you think all the green and red stuff left over at the end of refinement is "just noise", then I challenge you to re-collect your dataset and do the refinement again. You will find that the features "wiggle" a bit, but the difference peaks consistently pop up in the same place. "Noise" doesn't do that. But, if the only thing you are worried about is "validation" (aka "is my structure worse than average?"), then yes, once you get to the point when you can't find any way to build into the tallest difference feature you've got without making your R/Rfree worse, then you have reached the limits of current modelling technology and are basically done with building and refinement. Might as well deposit what you've got and wait for some future breakthrough to finally come up with an Fcalc that can explain your Fobs to within sigma(Fobs). -James Holton MAD Scientist On 6/26/2013 12:08 AM, [log in to unmask] wrote: > Dear Jiang Yan, > The Matthews function is based on an average protein crystal with 50% > solvent. However, crystals do exist with as little as 25% solvent or > as much as 75% solvent, so if your structure refines to an Rfree of > 20%, your structure is solved and you have a crystal with a high > percentage of solvent (which may explain your low Rfree). Maps are > usually contoured at sigma levels, based on the variation in the > electron density map. So with an Rfree of 20%, your difference map > will be very flat with and the sigma will be very low. Also the 3 > sigma level usually used for difference maps will be very low and > statistically, there are always some peaks at plus or minus 3 sigma, > but they are nothing to worry about. > Best, > Herman > > ------------------------------------------------------------------------ > *Von:* CCP4 bulletin board [mailto:[log in to unmask]] *Im > Auftrag von *?? > *Gesendet:* Mittwoch, 26. Juni 2013 06:05 > *An:* [log in to unmask] > *Betreff:* [ccp4bb] Rfree is 20%,why still green and red density? > > Hi everyone, > > I now meet some problems when trying to solve structure.Space > group is P6422, and Mathews function shows there are 4 molecules > in one asymmetry unit. However, Phenix-autobuild shows only 2 > molecules in on one asymmetry unit, after refinement, > Rfree=20%,(resolution is 2.8A). Although AA fit the map well, > there are still some green and red density. > I wonder if anyone met this problem before? Any suggestion is welcome. > > Best, > Jiang Yan > >