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I agree. Generally, if you see Fo-Fc density but no 2Fo-Fc density you will have a hard time modelling it with explicit atoms. You can play a bit with your solvent mask parameters to see if you can get rid of the density. If it is a real ‘hole’ in your protein, this is a great place for introducing stabilizing mutants. Cheers, Robbie Sent from my Windows 10 phone Van: [log in to unmask]<mailto:[log in to unmask]> Verzonden: woensdag 24 augustus 2016 09:16 Aan: [log in to unmask]<mailto:[log in to unmask]> Onderwerp: [ccp4bb] AW: [ccp4bb] Disappearing difference density? Dear Andrew, I suspect it is an artefact of the bulk solvent correction (or lack thereof in the inaccessible pocket). What is the absolute value of the difference density? Difference density gets by default contoured at +/- 3 sigma, so you will always get some peaks, even with almost perfect models. Best, Herman Von: CCP4 bulletin board [mailto:[log in to unmask]] Im Auftrag von Andrew Marshall Gesendet: Mittwoch, 24. August 2016 08:31 An: [log in to unmask] Betreff: [ccp4bb] Disappearing difference density? Dear all, I have a 2.6 A structure at the validation stage, stats look good (Rfree/Rwork : 25.7/19.7). I just have a patch of positive Fo-Fc density present in a solvent-inaccessible hydrophobic pocket which I'm struggling to fill. I've tried fitting glycerol (which seems unlikely, but it's the only component of the crystallisation buffer that seems about the right size) but the resulting Fo-Fc density is entirely negative (no 2Fo-Fc density). When fitting a water molecule I get the same result. I also tried the automated ligand identification tool in phenix which suggested a number of possible small molecules (all placed in this patch of difference density), including glycerol, but I get the same result after a round of refinement. Has anyone observed anything similar before? Thanks, Andrew Marshall PhD Candidate Laboratory of Protein Crystallography Dept. of Molecular and Cellular Biology School of Biological Sciences The University of Adelaide