Initialsing HSQCs

If  CcpNmr Analysis is not already open, start it up again 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.)

Open an existing project

In the Analysis menu bar select M:Project:Open Project.  Select [Yes] if Analysis asks whether it can close the current project and [No] if it asks to save the current project. Navigate to find and select the CcpnCourse1b project (the directory initially will be in green and then when you have selected it in deep purple). Then click [Open].

You might get a warning that various files have moved location. You might also get a dialogue box  with a list of spectra paths (because those also have moved location). If the paths are all in grey then just click the [All Done!] button at the bottom.  If any path is in red then Analysis cannot find the corresponding spectrum data file, so either you need to tell Analysis where it is (by double clicking the path cell and navigating to the correct location: CcpnCourse1b/data/dataStore1) or accept that that particular spectrum will not have its contours displayed.

This project has three windows, "window1", "window2" and "window3". The first is a two-dimensional window with an HSQC spectrum in it, and the other two are three-dimensional windows, one with an HNCA and an HNcoCA, and the other with an HSQC-NOESY. The HSQC has been peak picked but not the other spectra.

Assigning New Resonances

Given a project with some picked peaks we will start some assignment of those peaks to resonances. Initially we will assign the peaks anonymously, that is to say we will link peaks to a resonance number, but not say which atoms the resonances comes from. Initially such an assignment is not very useful, but we can go on to link related peaks to the same resonance numbers. For example we can say that a whole column of peaks in the HNCA and 3D N-NOESY and an HSQC peak are derived from the same amide resonances. When we specify which atoms the resonances derive from then all of these linked peaks will automatically be assigned to those atoms.

To start an assignment choose the isolated HSQC peak at  the location 7.27, 121.7 (1H,15N):

, and with the cursor near its centre press 'a' (or you can select R: Assign: Assign HQSC...). Note it doesn't matter which peak you choose really, but we will be referring to this one later. The Assignment: Assignment Panel popup will appear containing two rows of tables, one for each of the HSQC dimensions. In the left most table of each row click the button [<New>], this will add the resonances [1] and [2] to the 1H and 15N dimensions of the peak. Now click [Set Same Spin System] at the bottom left of the popup. The resonance assignments will become {1}[1] and {1}[2] here the {1} annotation signifies that the resonances are both in spin system number 1, which in this instance indicates that they both belong in the same amino acid residue:

Now mark the peak 'm' and navigate to the equivalent position in the 3D N-NOESY spectrum in window2 via R: Navigate: 1H - 15N in window3. Ensure the peaks along the marker in window2 are picked ('Shift' + 'Ctrl' + left click and drag):

Note that if you cannot pick some 3D peaks they may not have a maximum within the selected depth range. If this the case you can adjust the depth position or width via M: Peak: Peak Finding.

Now assign one of the 3D peaks at the marked amide location: Press 'a' with the cursor over the peak and find the Assignment: Assignment Panel popup. You will see that the popup has now updated for the 3D peak and consequently there are three dimension rows. Because the peak position closely matches the chemical shift value for resonances [1] and [2] they appear in the right hand tables. We can link these existing resonances to the 3D peak by clicking on their rows in the right-hand table. When you do this the resonance annotation appears on the left hand side (and in the spectrum window) to indicate that the peak is now linked, i.e. assigned to the resonances.

Note that you can remove the resonance assignment in the popup by selecting a row from the left hand side and clicking [Clear Dim Contrib]. The use of the term "Dim Contrib" here reflects the fact that a given peak dimension could potentially have a contribution from multiple resonances. So for example you can could have an ambiguously assigned NOESY peak where two different pairs of close resonances contribute to the measured peak intensity.

With the 3D peak's amide dimensions fully linked we will quickly give the other 3D peaks at the same amide position the same assignment. Do this by zooming out in the window so that you can see all the peaks along the marker line, select all the peaks including the assigned one (left click & drag) then select R: Assign: Propagate assignments. This will cause the resonances assignments {1}[1] & {1}[2] (displayed on the spectra as "{1}[1],-,[2]") to be spread appropriately across all the selected peaks:

Efficient Resonance Assignment

The linking together of related peaks in different spectra by assigning them to common (anonymous) resonances is something that can be partially automated to speed up the assignment process. Of course you can also do this manually, as we illustrate above, if you wish. We can use the HSQC positions to define unique amide locations and pick and assign related spectra based upon these "root" locations. The first step in this automation is to define new amide resonance and spin system identifiers for all the peaks within the HSQC spectrum. Select M: Assignment: Initialise Root Resonances.

When you see the Initialise Root Resonances popup, there is a table called 'Amide Sidechain Peaks' with a few rows filled. Some of the peaks in the HSQC will be from NH2 groups of amine side chains, and you need to handle those before you can initialise the peak list. Clearly the NH2 groups give two peaks, one for each hydrogen but both have the same 15N resonance (and thus 15N chemical shift). If such pairs of peaks were processed in the same manner as the backbone amide peaks they would become linked to two different pairs of resonances in two spin systems, when in reality they should carry the same 15N resonance and be in only one (side chain) spin system. Click on a row of the table to view the peaks, first making sure you set it to follow the right window (here "window1"). If you think this looks like side chain NH peaks, double click the 'Confirmed' column so it changes to 'Yes'. Hint: there are five NH2 side chains in this protein. When you are happy with all of them, click [Initialise Peak List!] at the top:

This command calls the anonymous resonance and spin system assignment routines on all of the HSQC peaks. Note that the routine knows which spectrum to work with because the experiment type of the HSQC is set correctly as H[N]. You will now see that all of the peaks carry assignments of the form {x}[y],[z]. If you look at the NH2 peaks that you confirmed, you will see that both peaks belong to the same spin system and that the 15N dimension is assigned to the same Resonance.

The next part of the assignment process is to link resonances from the HSQC to the corresponding trains of peaks in the 3D experiments. From the menu select M: Assignment: Pick & Assign From Roots. In the Pick & Assign From Roots popup that appears ensure that window1 is selected in the Root Window pulldown menu and select window2 in the pulldown menu in the Target Windows section, and click [Add Target Window:]. Repeat this again to add window3 to the target list: select window3 from the pulldown list and [Add Target Window] again:

Now take a quick look at the {Tolerances} tab and set the "Root 1H Dim1" tolerance to 0.03 ppm and the "Root 15N Dim 2" to 0.2 ppm. Check if the other parameters look OK, and go on to the {Link Peaks} tab.

You will notice that the peaks from the HSQC are listed in the table:

If you click on one of the rows, window1 will centre on that peak and the location of window2 and window3 will move to the same amide frequencies. Rearrange the positions of the windows so that you can clearly see all of them, and the popup. Select a row corresponding to an HSQC peak that is not overlapped and click [Pick & Assign Root Resonances]. You will see that peaks are picked in the 3D spectra, in the box defined by the tolerances, and assigned to the amide resonances from the HSQC. You can go on to further amide positions by clicking [Next Root]. With appropriately set tolerances you may also click [Pick All & Assign Root Resonances] to process all of the amide positions - you can still use the 'Next Root' function (etc.) to loop through the peaks afterwards. Note that closely overlapping amide resonances would still have to be checked or linked by hand.

It is important when picking peaks and assigning resonances in this automated manner that noise and artefact peaks are not picked. Of course any offending peaks can be deleted afterwards, but most can be avoided by setting the picking tolerances to appropriately small values and setting the contour levels so that the noise is not visible. By default the peaks are picked only above the visible contour base level.

Now that we have defined and linked many resonances, look in the main resonance table at M: Resonance: Resonances. You will see that all of the resonances are listed here and many operations can be performed on them:

The important thing to note here is that the chemical shift of each resonance is automatically calculated from the positions of the peaks to which it is assigned. Note that a resonance assigned to only one peak will have no deviation in its shift, but those assigned to several 3D peaks will deviate as the amide peak position varies slightly. By default the chemical shift values are the average of the assigned peak positions, where each spectrum is weighted equally. However, different dimensions of different spectra can carry different shift weightings (set at M: Experiment: Spectra, {Tolerances}) so that the value of a shift may be influenced more by the more precise experiments.

Data display tables

The standard data tables, like the Resonance table, have some useful features built in. Shift-click and Ctrl-click allows you to select sets of rows. You can sort on individual columns by clicking the column header. Clicking the right mouse button brings up a menu that lets you filter rows on their contents, graph one column against another, and export sets of columns in a tab-separated format for scripts (or Excel).

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