Thank you for valuable advice of the BB. Negative density is observed between sulfur atoms of the disulfide pair, and positive density at the opposite side of the SG atom. I guess it is partial reduction due to radiation damage. Now, I refined using a altlocs and disulfide_bond_exclusions_selection_string to avoid forming a disulfide by altlocs of SG (because the rotamer is the same that the disulfide pair). Around 70 % occupancy is obtained for reduced Cys, and distances between SG increased near to 3 A. Difference density mostly dissapeared. I suppose is better to model only as disulfide for deposition, because reduction is "artifactual" from biological point of view, but it is other discussion... Thank you!Ezequiel El Lunes, 15 de febrero, 2016 8:41:49, Pavel Afonine escribió: Hi Ezequiel, Sorry, my previous email was incomplete. My structure has negative density in the center of some disulfides (the magnitude of this effect vary between different monomers in the ASU). I'd like to refine the structure in an "artificial reduced form", where cysteines involved in disulfides are treated in reduced form, but repulsions originating from neighboring sulfur atoms are turned-off, to monitor distances (I expect that SG atoms remain close to 2 A), guided only by data. there is no option like "disable non-bonded repulsions in a sphere R=??? Angstroms around given atom". However, there are couple of tricks to do what you want, such as defining bonds between atoms in question with large sigma which will make them invisible to restraints and also disable repulsions. Or playing with altlocs. You can check .geo file to see exactly what restraints atom in question participates in. Pavel

If you're using an older version of Phenix with TLS refinement, this may be simply due to the issue of the radius for B-factor restraints being less than the length of the disulfide bond (so the two sulfurs aren't restrained to similar B-factors). Looking back at some of my older refinements, I see many disulfides with negative density blobs that are probably attributable to this. ________________________________ From: [email protected] on behalf of Pavel Afonine Sent: Monday, 15 February 2016 6:46 PM To: Ezequiel Noguera; [email protected] Subject: Re: [phenixbb] Update: Check for radiation-induced reduction of a disulfide bond - PS P.S.: If atom is misplaced normally you would see both: positive and negative residual density in close proximity to each other. If you only see negative density at SG centers this may mean the site is partially occupied. Pavel Sorry, my previous email was incomplete. My structure has negative density in the center of some disulfides (the magnitude of this effect vary between different monomers in the ASU). I'd like to refine the structure in an "artificial reduced form", where cysteines involved in disulfides are treated in reduced form, but repulsions originating from neighboring sulfur atoms are turned-off, to monitor distances (I expect that SG atoms remain close to 2 A), guided only by data. Thank you,