Generate restraint lists

Instructions

This HowTo will describe how to generate both dihedral and distance restraint lists that can be used for structure calculations.

Generate distance restraints from assigned peaks

To make a list of distance restraints from the assigned peaks, go to {Settings} in Structure: Make Distance Restraints accessible through M: Structure: Make Distance Restraints. Select a peak list for a spectrum with through space interactions (e.g. a 15N-NOESY-HSQC peak list) and set parameters.

• • The ‘Restraint Distance Params’ section allows us to specify how the peak intensities relate to the distance bounds of any generated distance restraints. The default method in “Distance function:” is to calculate a target distance as peak height raised to the power of -1/6 multiplied by some scaling factor, such that the reference intensity (in this case defaults to the peak list's average volume) exactly corresponds to the reference distance (in the default case 3.2 Angstroms).

  • The lower and upper bounds of the distance restraint, “Lower frac error:” and “Upper frac error:” are calculated as fractional changes from the calculated target distance (default is 20% for both).

  • The absolute minimum and maximum (“Lower dist limit:” and “Upper dist limit:”) values for the bounds are set to 1.72 and 8.00 Angstroms respectively by default.

To derive restraints from assigned peaks from the selected peak list simply click [Make Assigned Restraints]. The {Residue Ranges} and {Chem Shift Ranges} tabs allow you to make restraints only for specific residue regions of your molecule or specific shift ranges.

After some time you will see the Structure: Restraints and Violations popup appear. In the {Restraints} tab, you will see the restraints listed, mostly as green colored rows. Note that some restraints have grey rows. The grey rows indicate restraints that are ambiguous, e.g. a possible restraint between two different pairs of 1H resonances. Note that such ambiguous restraints can represent logical uncertainty (e.g. before an ambiguity is resolved) or real physical ambiguity, where a peak is caused by two or more overlapping pairs of resonances.

Generate distance restraints from unassigned peaks

There is a second common way to generate distance restraints. This is done by matching chemical shifts of resonances to NOE peak positions, thus generating potentially highly ambiguous distance restraints. One reason for making your own restraint lists in Analysis is that Analysis gives you more control of the process and is better for certain tasks, like handling aliased peaks. The {Shift Match Tolerances}, {Chem Shift Ranges} and {Network Anchoring} tabs in the Structure: Make Distance Restraints popup allow you to generate distance restraints for peaks which do not have assignments.

• • To generate distance restraints for specific assigned regions by shift matching, go to {Shift Match Tolerances}. Set peak list and other options and click [Make Shift Match Restraints].

  • To generate distance restraints for specific shift ranges by shift matching, go to {Chem Shift Ranges}. Set peak list and other options and click [Make Shift Match Restraints]. The “shift matching” method generates potentially ambiguous distance restraints by simply matching peak positions to close chemical shifts.

  • To generate distance restraints using network anchoring and shift matching, go to {Network Anchoring}. Set peak list and other options and click [Make Shift Match Restraints]. In the “network anchoring” method, chemical shifts are also matched to peaks, but the ambiguous possibilities are refined by selecting only NOE assignments among the possibilities that are supported by other assigned NOE’s or the covalent structure.

Generate dihedral restraints

To generate dihedral restraints from the backbone chemical shifts, use the program DANGLE (Dihedral ANgles from Global Likelihood Estimates) which is embedded within Analysis. DANGLE estimates dihedral angles from chemical shifts in a similar manner to TALOS; i.e. it matches a chemical shift & sequence query to a structural database of known PHI/PSI angles and chemical shifts.

To run DANGLE use M: Structure: DANGLE: Predict Dihedrals. This opens the DANGLE popup:

• • In ‘Options’ set the “Chain”, the “Shift List” and “Max No. of

  • Islands:” To specify which data to use and how strict the analysis should be. Using e.g. two islands means that we will reject predictions that result in more than two discrete regions in the Ramachandran plot.

  • To start the analysis click [Run Prediction].

Once the calculation is over you will see that the ‘Dihedral Angle Predictions’ is filled with PHI and PSI backbone dihedral angle predictions and their associated error ranges. If you select a row you will see a plot in Ramachandran (PHI/PSI) space of where the likely angles are deemed to be. A lot of red colour in the chart indicates that DANGLE was not able to make a distinct choice of PHI/PSI. You should not use such a prediction in a structure calculation. Residues that have a very precise range of predicted PHI/PSI angles can be used for structure calculations with a high degree of confidence.

To make a Restraint List of the Restraints:

• • Set "Constraint Set:" to which Restraint Set you want to add your dihedral Restraint List to.

  • Click [Commit Restraints].

The Restraints List is now accessible via M: Structure: Restraints and Violations, {Restraints}.