Multiple Shift Lists and Copying Assignments

If the program is not already open, start CcpNmr Analysis on the command line by typing:

-> analysis

(This assumes that the CCPN bin/ directory is on your path, otherwise you will need to type the full path or be in the bin/ directory.)

Setting up and linking a new shift list

So far during this tutorial, all of the chemical shift values we have recorded, by picking peaks and linking resonances & atoms, have gone into one single list. However, there are several circumstances when it is helpful to use more than one list of chemical shifts, so that you may for example separate sets of shifts based upon the experimental conditions they relate to; you may use different shift lists for different temperatures or when doing titration experiments. Commonly in such circumstances the underlying resonances (and hence atoms) that we work with are the same, but the positions of the peaks move significantly and we would not want to average chemical shift values over such a variation. In the Analysis menu bar select M: Project: Open Project. Select to close the exiting project, but there is no need to save. Navigate to find and select the CcpnCourse2c project, then click [Open].

We are going to set up a new shift list for the second HSQC experiment present in the demonstration project. This second HSQC experiment relates to exactly the same sample (molecule) as the first HSQC but was recorded at a different temperature, which has caused the peak positions to move. Given that the second HSQC experiment is already loaded, all we need to do is go to the M: Experiment: Experiments, {Experiments} and in the Shift List column double click on the cell corresponding to HSQC_2. In the pulldown menu that appears simply select <New>. You will see that a new shift list is now listed:

From now on whenever an assignment is made in HSQC_2 the shift remains separate from the other spectra. Indeed, any resonances that were already connected to HSQC_2 before its shift list was changed will have their shifts recalculated in two separate groups. You could move the experiment back to the original shift list at any time and the chemical shift values will be appropriately recalculated. However, if you disconnect experiments from a shift list (or delete a peak etc) you might have chemical shift values that are not defined by any peaks. Under such circumstances the chemical shifts persist, but are described as "orphans".

Assignment of HSQC_2, with its separately curated shifts proceeds pretty much as assignment normally does, with the only major difference being that for a new shift list (i.e. without any assignments under it) there will be no resonance possibilities that can appear when assigning a peak. - The only known shifts are distinctly different, representing different conditions.

To assign HSQC_2, even though we don't have shifts set, we can say that certain peaks in the two HSQC spectra are equivalent if we can see how they have moved under the different situations. Accordingly we can select a peak in the first HSQC and the corresponding peak in HSQC_2 and propagate assignments. That is to say that the peak dimensions link to the same resonances (and hence atoms), even though things have moved. Go to spectrum window1 and try this for any pair of close, isolated peaks; one peak in each of the HSQC spectra. Use the mouse with a left click to ensure that these peaks (and only these peaks) are selected. Then in the right mouse menu select R: Assign: Propagate assignments. Note that this function usually checks to ensure resonance positions are within tolerances, but here with the two shift lists it cannot so the connection is made anyway, which is exactly what we want.

You may like to repeat the above procedure for several HSQC peaks and their counterparts. With a few resonances represented in the new shift list (should be "ShiftList 2") have a look at the resonance table M: Resonance: Resonances to see evidence of these links. In the resonance table note that you can choose between the different shift lists in a pulldown menu at the top, i.e set the ShiftList: pulldown to "ShiftList 2". Note that when the shift list changes the resonance positions shift and the number of peaks linked also changes. Apart from the Resonance Table, Analysis also provides direct access to the shift measurements. Looking in the menu at M: Data Analysis: Measurement Lists, you will see that there are tables of the shift values corresponding to each list. Note that other types of value like T1 rates, or Hetero-NOE values will also appear as measurements in these tables if they are calculated.

Copying peaks between related spectra

Propagating assignments between peaks by manually selecting them, although sometimes necessary, can be tedious if you have large number of peaks that are equivalent and have closely matching positions. In this instance Analysis provides a utility to compare all of the peak positions in two related spectra and transfer the resonance assignments efficiently, whilst still leaving sufficient scope for human intervention. 

In this exercise we will transfer the amide assignments from the peaks in the HSQC experiment to the HSQC_2 experiment. In the main menu we select M: Assignment: Copy Assignments. In the Copy Assignments popup ensure that the first "HQSC:115:1" experiment is set as the Source Peak List and that the second "HQSC_2:48:1" is set as the Target Peak List:

In the top table you will see a list of the source peaks, together with an indication of how many destination peaks they match, within the given tolerances. Note that when the Follow Peaks? option is set clicking on a source row will cause peak to be highlighted in a relevant window (window1). Also, selecting the source will display the potentially matching destination peaks in the lower table. Clicking on a target row will, as before, highlight the relevant peak.

This utility can be used in one of two ways. One can manually choose the correct destination for each source peak and [Assign Selected Target], or if you trust the completeness and accuracy of the destination one can automatically assign the closest destination peak to each source one with [Assign All Singly Matched]. When taking the more automated approach it is common to start with strict tolerances to assign the more certain matches and then relax the tolerances to fill in the remainder.

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