Assigning root resonances (2)

Analysis has a very useful tool for automatically assigning 15N-HSQC peaks to resonances and grouping them into spin systems, without having to do all the actions in the previous step manually. This procedure is called initialising root resonances, and is called using the initialisation macro in Analysis. For a fresh restart, open the project NapD_3.

Initialising root resonances

Initialise the root resonances by using the flowchart at the bottom of this page. This is done in the Assignment: Initialise Root Resonances popup:

Before the peak lists are actually initialised, Analysis identifies possible NH2 peaks in the 15N-HSQC spectrum based on their nitrogen frequency. NH2's come in pairs of 2 peaks with the same nitrogen frequency and different hydrogen frequencies. Usually they are in the upper right part of the 15N-HSQC spectrum, and they have small satellite peaks located slightly above the main peaks. The satellite peaks are caused by the presence of deuterium in the sample, and help to identify NH2's manually (note that these satellite peaks have been omitted from the 2D peak list).

To start the identification of the NH2's, set the tolerance for nitrogen to 0.025 ppm and click [Update Table] (the default of 0.02 ppm will miss one of the pairs whose peak maxima are slightly shifted due to overlap). When you click on the possibilities listed in the table rows, Analysis navigates to- and marks the possibilities in the 15N-HSQC spectrum. When you identify the pair as originating from NH2's, double click the |Confirmed?| input widget for the selected row to set |Confirmed?| to "Yes":

When you are done identifying all the NH2's, click [Initialise Peak List!] at the bottom of the popup. In the 15N-HSQC spectrum you will now see all the peaks assigned to spin systems and resonances.

A lot of information is displayed now, and using the command R: Peak: Label Auto Arrange (shortcut: 'w') on selected peaks, you can automatically adjust the peak label positions to optimize the view a bit.

When you inspect the spectrum, all backbone NH peaks have been assigned to atom types H and N, whereas the NH2's have not been assigned to atom types (verify this).

The project at this point is saved as: NapD_4.

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