Restraints & Violations
This part of the Tutorial is based on the CcpnStructCourseD project and will briefly go through Restraints and Violations in CcpNmr Analysis.
In the Structure: Restraints & Violations popup (M: Structure: Restraints & Violations), choose Restraint Set "2" at the top; these are the results from the ARIA run and choose the {Restraint Lists} tab. You will see that ARIA has generated one or more restraint list for each peak list and restraint list that was used as input data. Choose the second row labelled "ARIA_REJECT_run1_NOEs_it8", which represents the peaks/restraints that ARIA rejected, and select the {Restraints} tab. From the Structure pulldown menu at the top check that "2" is selected (the water refined ensemble) and in the Violation List pulldown menu table select "1"; you will hopefully see that many of the rows become orange/red, indicating that these restraints caused violations:
If you change the Restraint List pulldown menu to a list that is not labelled as "REJECT" you will see that there are far fewer violations.
Choose a restraint row for one of the violated (red/orange) restraints and click [Show Peaks]. This will open a table containing the peak (or peaks) that gave rise to the restraint. Note that a restraint may be linked to more than one peak, for example where there are symmetry related peaks that correspond to the same close resonance pair. In this Selected Peaks table click on the peak row and then the [Find Peak] button at the top. You will be whisked to the point in the spectra where the peak resides. From here you may choose to look at the peak assignment by using the 'a' key with the mouse over the peak. You can get to the same point directly from the restraint by using the [Assign Peak] button from below the Restraints table. Note that ARIA has generated an assigned peak list, so when we look at the N-NOESY spectrum we see two, differently assigned, peak lists on top of one another. To switch off the new peak list in the spectrum window you can click on the [Peaks] button at the top and toggle the [N-NOESY:182:2] option:
Now go back to the list of restraints. Click on the heading of the |Mean Viol| column then scroll to the bottom of the table to see the largest violation. Select the last red row (probably restraint number 77) and click [Assign Peak]. In the Assignment: Assignment Panel popup that appears/updates you will see that the original peak that was used to make the restraint does not have an assignment an the second dimension. This peak was not satisfactorily assigned during the structure calculation process, but this is not surprising given that no care was taken in generating an initial set of seed assignments; we deliberately wanted a project with lots of interesting violations. For the second "F2" peak dimension in the Assignment Panel click on the |Dist| column (mid-right table) so that the possible assignments are presented in order of distance from the assigned amide proton. Hopefully, you will see that there are two resonances "3GluHga" and "3GluHgb", from the same residue as the amide, at 2.7 Angstroms and that the chemical shift difference to the peak position |Delta| is 0.044 PPM:
These options are close enough in both chemical shift and structural distance to be good assignment candidates, thus click on both the "3GluHga" and "3GluHgb" rows for the second dimension to assign them. The peak will now have contributions to both resonances.
Repeat this operation once more for the second to last restraint (number 22), again clicking [Assign Peak]. This time you will see that there is an assignment option for "36IleHa" which seems plausible. Although this is not the closest possibility in the structure ensemble (at least the what we have so far) it is chemical shift difference is fairly small, unlike the "41ProHdb" option. Once we have checked all of the rejected and seriously violated restraints, adjusting their peaks accordingly we would go through another round of structure calculation, with a better starting set of NOE assignments. Although we can go though each problematic restraint in a fairly manual matter, there is a more automated procedure for checking NOE peak lists and curating their assignments using chemical shift and structure information.
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