Hi everyone, I would like to have commnets from experts on the following issues. I am refining DNA-protein complex at 3 A resolution and my current Rfree is 21.5 % (phenix.refine). 1. I am getting difference map peaks (in Coot) of magnitude less than -5 sigmas (0.24 e/A3) (~10 or more peaks) and equal number of positive peaks of same magnitudes.The positive peaks hardly have 2fo-fc of more than 1.0 sigma level. I presumed this well could be due to bulk solvent or improper mask. Therefore I optimized the mask parameters (by giving option under "General refinement parameters" in phenix.refine GUI). I could get my R free lower as expected but I still got these peaks back. Although I am not absolutely sure, but positive peaks are more in polar pocket of protein and negative peaks are more in non-polar and aromatic pockets. I have PEG, Na acetate in my crystal soup. What these negative peaks represent for? 2. During addition of atoms like Na+, Cl- in map, do one need to careful about of the its coordination valency in surrounding pocket. ? I will be highly thankful to you for the same. Ravi Ravindra D. Makde, PhD Postdoctoral fellow, Tan lab, The Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA