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Dear all
I am refining the crystal structure of a protein (Rfree and Rvalue are <25
and <20 A respectively). However, I am getting the negative density at some
places in the side chain of residues. All side chains are properly fitting
into the blue density, however red density blobs are also present at the
same place along with blue density . At some other place this red density
is also present in the main chain along with blue density (see the attached
snaps). If I have mutate the residues to alanine then density becomes blue,
but when change into the original residue, after refmac5 again it is
showing red blob. Also if I rotate the chain to place it in green density,
but after running refmac it attain original position having red blob. I am
not using TLS.
I am seeking some strategy so that the problem can be solved. Please
suggest me the possible reasons and remedy. Also i am naive in
crystallography.

Thanks
Alaksa