Basic Peak Picking & NOE Assignment

Window Components and Views

Once the new spectrum has loaded it will automatically appear in "window2". This is a three-dimensional window with "1H" along the x and y axes and "15N" along the z axis.  A new window is created like this only if no existing window matches in isotopes.

Note that if you minimise or close a window you can get it back again by going to M: Windows: HN: window2 (or equivalent):

You can move around the spectra within the windows using several different inputs. To zoom in and out use the 'PgUp' and 'PgDn' keys, the middle mouse wheel (if you have one), or hold 'Shift', click the middle mouse button, and move the mouse up and down. If you zoom out from your spectra so that you can see their edge you will notice a dotted line which denotes their border.

You cannot zoom out further than the maximum size allowed in each dimension. To change this size open up M: Window: Axes and select the {Axis Types} tab (if it is not already selected). Each Axis Type has a |Region|, which can be edited by double clicking on the relevant cell.

To pan around the spectra you can click and drag the scrollbars at the edges of the windows, use the arrow keys, or click and drag on the spectra with the middle mouse button.

Looking in "window2" move the extra scrollbar at the very bottom of the window. - Left click and drag. This scrollbar is present on the 3D window to change the depth or plane of the spectra being viewed. On a 4D window there would be yet another scrollbar. To change the thickness of the displayed planes click and drag the side of the depth slider with the middle mouse button.

Staying in window2, click on [Spectra] at the top of the window. Here you will see two coloured buttons, one for each of the 3D spectra. If you click the buttons you can independently toggle the contour displays for the two spectra on and off.

Setting Contours

To change contour settings click on the [Contours] button at the top of a window.  This lets you do simple adjustments.  The green arrows will raise or lower the contouring floor, while the +1/-1 will change the number of contours. The [Pos/Neg] button allows you to swap between showing just positive or just negative or both contour levels. Note that using these contour options only changes the spectra that are visible at the time within the window. If you click on the [More..] button you will bring up a more detailed contour level setting dialog.

Peak Picking and Manipulation

Focusing on the newly loaded N-NOESY spectrum the next task is to define some contour extrema as peaks. Note that peaks will be picked for all spectra that have their peaks displayed in the window. You can avoid that by turning off display of the spectrum (in [Spectra]) or peaks (in [Peaks]). There are two common ways to pick peaks. One is to search for all extrema in a boxed region. Try this in "window2" for the new spectrum by holding down 'Shift' and 'Ctrl' whilst clicking with the left mouse button and dragging the box to define a rectangular pick region. If a peak is not easily picked in the 3D spectrum, it is possible that the current Z range (depth) of the window view does actually cover the peak center. This is easily adjusted by using the bottom-most depth scrollbar.

Note that when the peaks are picked the contours will carry diagonal crosses to denote the peak position. Each peak will also have some annotation to the top right of the cross, which we discuss below.

Locate a point where the contours represent two overlapping peaks where the extrema search will only pick one of the two underlying signals. To define the second peak position hold down 'Ctrl' and click on the location to pick the new peak, although the user should be careful to ensure that the current Z (depth) position is correct. The R: Locate Peaks: Center z planes on peak menu option can be helpful to bring an existing peak to the centre of the depth view. Also, it should be noted that manually adding a peak at a specific position, i.e. not looking for signal extrema, will add in all visible spectra. Hence, if you wish to pick in only one spectrum, all other spectra should be toggled off.

Now we will select some of the peaks, e.g. artefacts or noise, for deletion. To delete peaks click with the left mouse button and drag a box over a region containing peak crosses (without holding down any keys). When the mouse button is released you will see that the peaks in the defined region are highlighted with a border around the cross. To select just a single peak click near its centre with the left mouse button. To delete the selected peaks press 'Del' or select R: Peak: Delete selected.

If you have peaks selected and then select a different set of peaks you will see that the selection is completely substituted for a new one. You can add to an existing peak selection by holding 'Shift' while you choose. Note that you can select peaks in several different spectra, and from different windows, in this manner.

Markers and Navigation

Pick and select an isolated peak in the HSQC spectrum (window1). Put a mark through it by holding the cursor over the peak centre and pressing 'm'. The lines produced are a multi-dimensional marker at the peak position and will be visible at the equivalent 1H-15N location in the 3D window. To go to this equivalent position in window2, with the cursor over the marked peak select R: Navigate: 1H - 15N in window2. Note that window3 has HHN axes and thus has two navigation options, where the second option would take you to the 1H position on the vertical axis, rather than the horizontal axis, which represents the amide proton.

Multi-dimensional marks, vertical ruler lines and horizontal ruler lines can be added to any window location, not just on peaks, using the 'm', 'v' and 'h' keys respectively. To increase the number of marks and rulers that can be displayed at one time select M: Window: Marks and Rulers or R: Markers: Options. Note that you can clear all marks and rulers with the 'n' key.

Strips and Strip Navigation

Now we will start to manipulate "strips" which are sub-divisions of a window that are connected (in terms of their view) in one of the screen dimensions, but independent in the other dimensions. Go to window2, select the "Strips" option at the top and click [+], this will add a vertical division to the window. Click and drag with the middle mouse button to move the spectra - you will see that the vertical axes of the strips are tied together, but the horizontal axis is independent. The depth dimensions are also independent, e.g. if you move the bottom most, 15N scrollbar. Which depth dimensions are moved depends upon which strip is active. The active strip is indicated by an asterisk "*" next to its strip number and is set either by double-clicking (left mouse) within a strip or by using the strip options at the top of the window; by clicking [1] or [2] or whatever. The active strip is also the one that will be removed when the [-] button is clicked. You can rearrange the strip order, moving the selected strip with the green arrows, and swap between vertical and horizontal strips with the toggle button. To remove all the strips press the buttons with vertical and/or horizontal stripes to the right of "Clear:".

Manually locating strips at interesting positions can be tedious, but there are various options to build strips from pre-defined locations, for example using peaks. To make strips using peak locations select three (picked) peaks in the HSQC spectrum and select (using the right mouse button) R: Strip: Peak Location Strips:1H-15N in window2 (the same option is found in R: Navigate). The result is three strips located at the amide positions corresponding to the HSQC peaks.

Manual Resonance Assignment

Once we have some picked peaks we can assign those peaks to resonances. Given that we are starting from a CCPN project where the resonances have been almost fully assigned to atoms, what we need to do to the N-NOESY spectrum is link its peaks to resonances that have already been identified, and naturally are close in chemical shift to the peak position. For example we can say that a whole column of peaks in the 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 43Val HSQC peak at  the location 7.27, 121.7 (1H,15N). Mark the peak 'm' and navigate to the equivalent position in the 3D N-NOESY spectrum in window2 via R: Navigate: 1H - 15N in window2. 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:

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 43ValH and 43ValN 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:

If there was a unique match for the second 1H dimension we could also assign it at this time, however most peaks will have a number of ambiguous options and we can let a structure generation and NOE assignment program like CYANA or ARIA resolve the full assignment.

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 43ValH and 43ValN (displayed on the spectra as " 43ValH,-,N") 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 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 the amide locations and pick and assign related spectra based upon these "root" locations. 

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:]. Make sure that both N-TOCSY and N-NOESY are set to Active:

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  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. Try it. You can still use the [Next Root] button (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 artifact 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 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:

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