Restraints

Introduction

The penultimate part of the exercise is to look at how we can use Analysis to generate distance restraints that may be used in a structure calculation and how we can pass intermediate structural information back into Analysis to help with NOE peak assignment.

Distance restraint lists

To make a list of distance restraints from the assigned peaks in an NOE peak list first go to M: Structure: Make Distance Restraints:

At the top of the popup, change the peak list to "N-NOESY:182:1". We can leave all of the other parameters alone for demonstration purposes. The 'Restraint Distance Params' section would allow us to specify how the NOE peak intensities relate to the distance bounds of any generated distance restraints. The default method is to calculate a target distance as peak volume 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 this case 3.2 Angstroms). The upper and lower bounds of the distance restraint are calculated as fractional changes from the calculated target distance (default is 20% above and below) while observing absolute minimum and maximum values for the bounds (1.72 & 8.00 Angstroms respectively by default). The {Residue Ranges} and {Chem Shift Ranges} tabs would allow you to make only restraints for specific assigned regions of your molecule or for specific shift ranges.

Making restraints from assigned peaks

To calculate restraints for assigned peaks from the selected peak list simply press [Make Assigned Restraints]. After a short pause you will see the Restraints and Violations popup, {Restraints Lists} appear. This shows that you have one restraint set (a way of grouping related restraints and violations) containing one list of over 500 restraints. Click on the row of the restraint list in the central table and then click [Show Restraints]. Note that you can also get to this point via M: Structure: Restraints and Violations, {Restraints}:

The restraints popup will appear and in its table you will see the restraints listed, mostly as green coloured rows. Note some restraints also have following grey rows. These grey rows indicate restraints that are ambiguous, i.e. a possible connection between two different pairs of 1H resonances. Note that such ambiguous restraints can represent logical uncertainty (before an NOE is resolved) or real physical ambiguity where a peak is caused by two or more overlapping pairs of resonances.

Making restraints from unassigned peaks

There is a second common way to generate distance restraints, which is to match the chemical shifts of resonances to NOE peak positions, thus generating potentially highly ambiguous distance restraints. Such restraints would typically be filtered to select only the correct contributing resonance pairs, by iterative structure generation and violation analysis in a program such as ARIA or CYANA. Accordingly the {Shift Match Tolerances} and {Network Anchoring} tabs in the Structure: Make Distance Restraints popup allow you to generate such distance restraints for peaks which do not have assignments. To generate distance restraints by shift matching, click [Make Shift Match Restraints] This command uses the current settings, but {Chem Shift Ranges} and {Shift Match Tolerances} are only relevant for this command.

In the case of the shift-matching method potentially ambiguous distance restraints are generated by simply matching peak positions to close chemical shifts. In the case of network anchoring method chemical shift are also matched to peaks, but the ambiguous possibilities are refined by selecting only NOE assignments from amongst the possibilities that are supported by other, assigned NOEs or covalent structure. Say, for example, that a peak could arise from a number of resonance pairs. Two resonances A & B are more likely to be a correct assignment for the peak if we know that they are close to (or bound to) the same intermediary resonance, C and therefore must be close to each other.

Restraint sets

Note that the restraint set to use is defined in the Structure: Restraints and Violations popup, in the {Restraint Sets} tab. Most of the time you would use the same restraint set as was used previously. You always have the option to put new restraints in a new restraint set, and indeed you should always use a new restraint set if any of the atomic assignments have changed. Each restraint set uses a frozen copy of the resonance-to-atom assignments at the time its restraints were created, so that you always have a record of what was really restrained, even if the resonances subsequently are reassigned to different atoms. By the same token, if you did not use a new restraint set after atomic assignments were changed the restraints would refer to the atoms from the old assignments not the new ones.

In the Structure: Restraints and Violations popup, which is hopefully still open (M: Structure: Restraints and Violations otherwise), choose the {Restraint Lists} tab and select restraint list 2 (this was made from unassigned peaks) in the table and click [Show Restraints]. You will now see that in contrast to the first list these restraints are highly ambiguous, with several possible resonance pairs for each restraint:

If you click on the row of the first restraint "1:0" and then [Assign Peak], the Assignment: Assignment Panel popup will show assignment possibilities and status for the peak that gave rise to this restraint. This illustrates that in the second dimension of this NOESY peak there are several resonances with shifts that closely match the peak position for that dimension. Note that the peak itself is not assigned to any of these. The correct contributing resonances may be assigned after a structural model had been calculated. Referring back to Structure: Restraints and Violations, if you now click [Show peaks] you will see that the NOE peak appears in a table from where you can then select the peak's row and click [Find Peak] to locate it in an appropriate window. 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.

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