Hi.. I am doing real space refinement on a protein model build from a cryo-em map. I run the refinement with Amber gradients turned on and with secondary structure restraints (otherwise, more or less default settings). I have some issues with helix and sheet outliers. Every time I run the refinement (after making sure that the bond distance in the helices are within the 3.5 Å) I get a lot of bad annotation remarks, but with outliers just above 3.5 Å (see .log below): ... removed outlier: 3.576A pdb=" N VAL A 179 " --> pdb=" O LEU A 175 " (cutoff:3.500A) Proline residue: A 180 - end of helix removed outlier: 3.681A pdb=" N PHE A 188 " --> pdb=" O LEU A 184 " (cutoff:3.500A) removed outlier: 3.628A pdb=" N PHE A 189 " --> pdb=" O ILE A 185 " (cutoff:3.500A) removed outlier: 3.521A pdb=" N ILE A 190 " --> pdb=" O ALA A 186 " (cutoff:3.500A) Processing helix chain 'A' and resid 219 through 228 removed outlier: 3.737A pdb=" N MET A 227 " --> pdb=" O GLU A 223 " (cutoff:3.500A) ... Is this something that I just need to fix in several rounds of refinement or is there an explanation? Amber gradients maybe? In addition, I have some bad sheet annotations such as: ... removed outlier: 6.987A pdb=" N VAL B 433 " --> pdb=" O LEU B 440 " (cutoff:3.500A) removed outlier: 4.659A pdb=" N VAL B 442 " --> pdb=" O LEU B 431 " (cutoff:3.500A) removed outlier: 6.296A pdb=" N LEU B 431 " --> pdb=" O VAL B 442 " (cutoff:3.500A) removed outlier: 5.062A pdb=" N ALA B 444 " --> pdb=" O THR B 429 " (cutoff:3.500A) removed outlier: 6.129A pdb=" N THR B 429 " --> pdb=" O ALA B 444 " (cutoff:3.500A) ... When I look at the model, the residues are definitely a part of the sheet - the long distance between the N-O atoms is due to the O being "flipped" (hope it makes sense). Is this because of wrong annotation? Best Casper