Index:
The most important facility to understand in the Pronto program is the contouring facility. If you spend some time in the beginning by learning the different facilities in the contouring setup, you can save much time later on!
The main setup window for the contouring is shown in Figure 1. This is a rather complicated window, as the contouring setup should have facilities for plotting different spectra, plotting multiple regions of the same spectrum, and for displaying regions from different planes of a 3D spectrum.
The browser at the top of the window is used to save and recall different setups. The setups are stored inside the database, and will thus survive from one day to another. Pressing Save stores the current setup (the spectra selected and the regions to be displayed) under the name entered in the text field below the browser, in this case "CNT Name Unknown". A setup is loaded by selecting it from the list and pressing the Load button. The Delete button deletes the currently selected setup, but does not change the current setup. The Init button hasn't really anything to do with the saved setups: It resets the setup to the default values, leaving the saved setups untouched.
The browser in the middle of the window is used to select the spectra to be contoured. Currently, a maximum of eight spectra can be contoured. To add a spectrum to the list, highlight it in the spectrum window, and press the Add Spectrum button above the browser. The spectra in the list can be selected by clicking the left mousebutton with the mousecursor on top of the spectrum. The currently selected spectrum can be removed by pressing the Remove Spectrum button. The Zoom Spectrum button opens a window with the contouring parameters used for this spectrum - more about this later.
The bottom part of the window contains fields used for defining the range, or ranges, to be contoured. The example shown on Figure 1 is applicable to 2D spectra, only. The two scrollable lists define the chemical shift ranges in the x- and y-dimensions, respectively. The first column enables the range on that row. The second (empty) column is currently unused. The third column defines the center of the chemical shift range, and the last column defines the width of the chemical shift range, in ppm. Thus the ranges shown will display a contour diagram with an x range going from 8 ppm to 7 ppm and a y range going from 4.5 ppm to 3.5 ppm.
The contouring parameters used for the display of the TOCSY spectrum is shown in Figure 2. Five contour levels, from 2 to 10, will be displayed. Only the positive contours will be shown. If the "Lines: Positive" button is toggled to display "Lines: Positive Negative", also the contours from -10 to -2 will be displayed. The contours are drawn at equidistant intervals, as Lin/Log/Exp is set to Lin. The Color, Style, and Width define the appearance of the positive and negative contours. The meaning of the three marker types will be described later. A baseline correction can be applied by toggling the Baseline Corr. field to On. The next line defines the order of the Legendre polynomium. In this case, a second order polynomium will be used. The X, Y, Z, and W parameters define the permutation of the spectrum axes to be shown along the X, Y, and in the case of 3D/4D spectra, Z and W axis. The Z and W width parameters will be described later.
The table at the bottom of Figure 2 is the setup for the removal of overlapping cross peaks. This works as follows:
When the contouring is done, Pronto will remove the A cross peak part from the display by simulating this peak from table above. The B peak can now be added to the database.
The setup shown in Figure 1 and in Figure 2 generates the contour diagram shown in Figure 3. The meaning of some of the buttons in that window will be described below.
If Show Crosshair is enabled, a crosshair will follow the mouse cursor. The chemical shift values at the cursor position are shown at the bottom right of the display, always in ppm. The Focus Peak, Add Peak, Delete Peak, and Move Current Peak define the action taken when the left mousebutton is pressed when the cursor is in the contour diagram. The list of digits below the contour diagram, 1, 2, 3, etc. defines which of the possible eight spectra contoured is the current spectrum: The mouse functions in the contour window all work on the current spectrum only. If Focus Peak is selected, the peak closes to the position clicked in the current spectrum will be selected as the current peak. The peak selected will be highlighted in the cross peak window, if one is opened.
Peaks located in the spectra contoured will normally be labelled with crosses. A solid cross will be displayed, if the peak is used, i.e. assigned to one or more atoms. The cross for the current peak will be drawn using thick lines.
The setup shown in Figure 1 is typically used for locating peaks in a spectrum: You contour the spectrum in 1x1 ppm areas, locate the peaks in the diagram, go on to the next 1x1 ppm area, etc. You can use the arrow buttons above the range lists to step the center of the contouring ranges by one-half of the width of the range. You should never contour a whole spectrum, as this can take up a lot of memory, and can take a lot of time. There is currently no way of interrupting the contouring process. The program remembers the contours for the last 32 (can be changed in the resource file) contoured areas, so if these are 12x12 ppm, they can take up a lot of memory. The contouring cache for a particular spectrum is cleared, if you press Update in the spectrum zoom window, see Error! Bookmark not defined..
Some other typical contouring setups are shown in the next sections.
Instead of plotting large areas of a 2D spectrum, more detailed plots can be obtained by cutting up the spectrum into small fragments. Each fragment only shows a small (e.g. 0.2 by 0.2 ppm) part of the spectrum. This is done by enabling more than one region along the axes. This can be done fully automatic by the program, if you want to contour out the relevant peaks belonging to a spin system: Select a spinsystem from the spin system window, hold down the Set button in the contouring setup window, and select the Spin System entry. The chemical shift values relevant to the spectrum or spectra contoured are transferred to the range lists, and the range of the regions is taken from the Set Width parameter for that axis. An example of that is shown in Figure 4.
This setup produces a 3-by-3 contour diagram, where the center of each of the regions corresponds to the chemical shift value of each of the three protons in this spin system, in this case a spin system of an alanine.
The result of pressing Make Contouring is shown in Figure 5.
By adding a new spectrum to the list of spectra contoured, it is easy to locate peaks belonging to a specific spin system.
The result of adding a NOESY spectrum to the setup in Figure 4. is shown in Figure 6. It is thus easy to check for additional inter-residue effects.
Plotting of 3D spectrum on the screen requires a solution to the lack of dimensions available (2) on the screen. In literature and during lectures, 3D NMR spectra are often shown as "real" 3D objects, rotated according to a given viewpoint, and projected into two dimensions. However, this way of presenting 3D spectra is not very useful in the analysis stage. We prefer plotting 2D contour diagrams of planes taken out of the 3D spectrum. In Pronto this is done by adding the z axis along either the x- or the y-axis. The meaning of that is demonstrated schematically Figure 7. To the left, the chemical shift values of the z axis follow the chemical shift values of the x axis, i.e. each column is plotted from different planes of the 3D spectrum. If you prefer to have the windows in each row having the same z ppm value, this can be done by selecting the Z-by-Y option, as shown schematically on the right of Figure 7. Similarly for 4D spectra, each column can contain a contour diagram with different z value, and each row a different w value.
The corresponding contour diagram is shown in Figure 9. The spectrum plotted is a 15N-HMQC-TOCSY. The z axis is the 15N axis. The y axis has signals from protons attached to a nitrogen atom. Note that some of the apparent cross peaks are not marked with a cross. This is because the spectrum is transformed using a line broadening window function, resulting in peaks being several planes wide. Some of the "peaks" are thus just tails of peaks having a maximum one or more planes away from the plane plotted. The Z parameters in the contouring zoom window (see Figure 2) defines the width of the search in the cross peak catalog for the candidates for markers on the spectrum plotted. If Z width for Markers is specified as 1 ppm, peaks within ± 0.5 ppm of the center z value are labelled. The width specified for the z range defines the number of planes to display simultaneously. If a value of 3 (in Index units) is specified, three adjacent planes are added before the contouring takes place. It is thus possible to use a different width for contouring than for the display of markers.
The arrows on top of the z ranges can be used to step one half planewidth up or down. Setting the plane width to 1.0 index value and pressing an arrow twice thus steps from one plane to the next.
By simultaneous display of three adjacent planes it is very easy to see if a peak displayed actually has a maximum in the center range. An example for a setup for that is shown in Figure 10, the result in Figure 11.
Setting the chemical shift values from a spin system produces a setup like Figure 12. The ranges in the y-z dimension are merged, if both the y and the z ranges overlap. The width of the y range is used to determine if the y ranges overlap, the value of the parameter Z width for Merge (see Figure 2) is used to determine z range overlap. The values for Z widths are always specified in ppm. Similar widths exist for the W axis.
The result of plotting a spin system from a 3D spectrum is shown in Figure 13.
Two spin systems can be plotted simultaneously. By plotting both a TOCSY and a NOESY spectrum, two spin systems can be checked for inter-residue NOEs in a quick way.
The result of plotting two sequential spin systems is shown in Figure 15. The top spin system is Ile-76 and the bottom is Ala-77. The contours for the NOESY spectrum are drawn in green. Effects from amide proton of the alanine to the alpha proton of the previous residue can clearly be seen (at the crosshair position).