Reply To: Using Degenerate Protons in Rosetta3.x

[Anonymous]

I see. Due to the lack of documentation I thought that AmbigousNMRConstraint may be only used in the way I posted (because I found it in different NMR-related Rosetta literature).

I added the # sign to account for the degeneracy of NMR-derived proton constraints. In my spectra I can’t distinguish between hydrogen HG11, HG12 or HG13 of a methyl-group of a valine, for instance. It rotates so fast that any putative difference is averaged. Therefore, I only obtain the distance between the mean position of all three methyl-group hydrogens and whatever other proton from my NOESY-spectra.
There are two ways to deal with this in structure calculation software that I am aware of: a) averaging the hydrogen coordinates or b) adding some bias and take the heavy atom those hydrogens are bounded to (like described in the old Rosetta2.3 NMR-Guide). a) seems more accurate but given the fact that distances from NOEs are quite error-prone this 0.3-0.5 A offset is usually insignificant (unless NOE-derived distances were calculated quite laboriously from different experiments and including quantum-mechanic approaches).

What would be your suggestions for the following points:

1) Shall I format my constraints before feeding them to Rosetta to relate only to heavy-atoms to account for the proton degeneracy? This seems easier to handle than to make ambiguous constraints for each group of degenerate protons.

2) Since AmbiguousNMRConstraint is evaluated in the same way as AmbiguousConstraint I understand now why quite a lot of constraints seem to be neglected. Would ATOMPAIR in combination with the BOUNDED function be better? Until now I only tested ATOMPAIR + HARMONIC which, following your advise, is definitely the wrong way to handle this.