A recent structure at 2.5A was solved by our group. We observed that at the proposed peptide binding site extra densities (after Refmac) are present on 2Fo-Fc (1 sigma, same below), but interestingly not on Fo-Fc (3 sigma, same below) map. We therefore struggle to conclude anything from this. So is it an acceptable practice to build the peptide based on 2Fo-Fc? Also, would it be our data reduction / MR which has gone wrong which makes the two maps so different?
Of note, we deliberately deleted the N- and C-termini residues in our input model for better MR solution. After MR, the termini of the output model also show similar feature: electron densities on 2Fo-Fc but not on Fo-Fc.
Assuming that you removed all ligands and water from the MR model, your 2Fo-Fc map positive difference density is probably real. The peptide might also be present in a fraction of the molecules in the crystal form resulting in partial occupancy. Partial occupancies are easily refined in PHENIX. I suspect that if you increase the noise in the Fo-Fc map contour you will start to see the suspected peptide ligand (I sometimes go as low as .7 sigma for insight). It is important to refrain from modelling the peptide ligand into difference density until the MR solution is well refined with the completed N and C termini. I always save and file my map and model just before modeling the ligand to make sure that I can go back to it at anytime. Most crystallographers like to see the unmodeled 2Fo-Fc contour with the ligand placed (but unrefined) to show how well the ligand fits into the density.
If I were you I would complete the N and C termini, refine, examine the suspected binding pocket for positive difference density, save and file for future reference, build into the positive difference density and refine. Keep an eye on R and Rfree to ensure you are not over interpreting your map or over refining the model. What is your Rmerge? Completeness? Does the 2Fo-Fc density look like a peptide backbone?
Good luck.
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