Hi Alexandra,

1) I have a metal coordinated to a cofactor in the active center of my protein structure. The initial solution provided by phaser/autobuilding already contained the metal+cofactor modelled in the density but with zero occupancy. Then I created all the necessary restraints and cif files with phenix_ready_set and did two cycles of model building/phenix refinement. The refined structures always contained zero occupancy for those atoms. My question is: should I manually change the occupancy for the metal+cofactor to 1? I thought this would be made automatically by phenix because I selected the option “occupancies” and “individual B-factors” in the refinement strategy…

this is explained in the manual: https://www.phenix-online.org/documentation/reference/refinement.html#occupa... Please let me know if it is still not clear. In a nutshell, if you want occupancy to be refined automatically it needs to be between 0 and 1 in input PDB file.

2) When manually placing waters in the density with coot, what is the maximum value of B-factor that is acceptable for a water? I am asking that because I have some water molecules with weak density and consequently B-factors between 45 and 55 but that are involved in interactions with other molecules. Is it better to keep those waters or remove them?

No need to do it manually. Let phenix.refine update water (add/remove/refine) and check them once or so at the end of refinement to make sure all water make sense. There are way more considerations that go into decision-making about placing a water than just B-factor; typically they are considered all together. Pavel