Hi,

Supposed by phenix.real_space_refine I got a PDB of protein-ligand, will you please let me know how to get the subtraction map (difference map?) which only show the density of the ligand, so that I can see whether the ligand was well resolved in the map, or whether the built ligand fits well with the ligand density?

it's not yet available (assuming you are asking for this to be done in real space). However you should be able to use your original cryo-EM map to infer this information. Another alternative (which I can't dislike more) is to convert cryo-EM map into structure factors, run a round of phenix.refine refinement, and that will give you an MTZ with 2mFo-DFc and mFo-DFc map coefficients (which, by construction, will be model biased, unlike your original cryo-EM map!). How much sense they would make in this case that I can't tell. Pavel