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