Hi Wei,

I think I replied to a very similar question on this mailing list some time ago (in August).

There are two commonly used options for omitting the ligand in order to demonstrate its presence/absence in Fo-Fc OMIT map:

1) Physically remove the ligand from PDB file. Then do some refinement and calculate Fo-Fc map.

2) Keep ligand in the file, set its occupancy to zero. Then, again, do some refinement and calculate Fo-Fc map. In this case you may want to ask refinement program to not move the ligand or move it only a little.

Now, here is why these two options are poor and will not give you what you want.

In the first case the bulk-solvent mask will be set in the ligand region and therefore it will mask ligand density (bulk-solvent will be filled into the ligand region). Depending on the strength of ligand density it may be masked completely or deteriorated.

If you follow the second option you will always get positive density in ligand area. This density may correspond to bulk-solvent, ligand or mixture of both. That is there will be no simple way to differentiate whether this density arises from the ligand or bulk-solvent.

At some point I've spent a day doing tests to exemplify all this.

Here is how to do this right. You need to define a box around the ligand that covers the ligand itself and some volume around it, then omit all scattering from inside the box (ligand and bulk-solvent!), and then use this model with box omitted to calculate residual synthesis.

I don't think we have a ready to use tool in Phenix to do this. It's possible to do this with some scripting using CCTBX.

Pavel

On 9/13/14 2:40 PM, Wei Shi wrote:
Hi all,
I am working with a protein dimer-ligand structure (resolution: 2.8Å) and am going to make a figure showing the bound ligand superimposed with the electron density (Fo-Fc) map obtained after omit refinement.
What I did is to make the ligand-free structure model and then use Phenix.refine using the following parameters plus simulated annealing (torsion angle) or (Cartesian):
Default: XYZ coordinates + Real-space+ Individual B factors + Occupancies.
Also click: Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS.

Contoured at 2σ, the Fo-Fc map generated show green density for most part of the ligand, but for several atoms in the middle of the ligand, the green density is missing... It's like this: some atoms have green density and the next few no green density and then a couple few has green density and then a couple more no green denisty.... The missing green density usually means that part is disordered in the structure, right? I am not sure why the density is missing for some atoms in the middle of the ligand. It would makes more sense if the missing green density is at the end of the ligand...

Does anyone happen to have any clue about what it means for the green density missing in my case?

Also, I am not sure whether I did the omit refinement right:
(1). For simulated annealing, it has two options, one is Cartesian and the other is Torsion angle, and it's said that torsion angle is more suitable of low resolution data. Is there any difference about which one to choose in my case?
(2). Is it Okay to run the omit refinement in the presence of the following parameters as I did:
Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS.

Thank you so much!

Best,
Wei 



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