Water in protein structure modeling

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parkj77's picture
Water in protein structure modeling

I am new in the field of crystallography, and I have just started learning how to use Coot and CCP4i. I am experiencing difficulties dealing with water molecules in my structure. My questions are:
1. Coot detects unmodeled blobs (densities) that are too big for water in my structure. How do I know if these are actually something else and not multiple water molecules H-bonded to each other.
2. In some of the previously solved structures related to my protein, the depositors have modeled "Unknown ions" into their structures. If they do not know what these ions are, how do they know if they are not water molecules? How can one distinguish a water molecule from metal ions such as magnesium or sodium?
3. Using Coot, I manually modeled in water molecules into positive densities (green, or Fo-Fcalc). However when I ran refmac using CCP4i (restrained refinement) with the revised model, the R-free went up as the R-factor went down. Does this mean that whatever I have modeled in should not be there? Why does this happen when the map clearly suggest that something should be there?
Thanks a lot for the help in advance!