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SPSCAN Tutorial

HOW TO ...

Run the program:

related topic: how to handle the interface

Command line options

SPSCAN supports the following command line options:
-m macroname
Start handling the macro macroname. Continue normally.
-x macroname
Start handling the macro macroname and exit after completion of the macro.
-h macroname
As above, and hide all windows.
I cannot recommend to use this option: If the macro needs any interactive input the program will end up in an infinite loop, because it expects input which cannot be given. However, "-h" it is a way to run long macro jobs "nohup" in background.
Suppress automatic loading of a project file, although such a file is declared in the resources.
Enable menue for "project" - commands (sequential connection, automatic creation of proton list, mapping ...) in executables where this menue is disabled by default.

Integrate a 2D spectrum:

Integrate a series of 2D spectra:

Adapt ppm or not?
Within a series of relaxation experiments the linewidth of the peaks should not be changed. Adaptation of the exact position of the peaks, however, can be useful.
If ppm is not adapted, the volumes in the other spectra can be systematically underestimated when the peak is shifted away from the position in the reference spectrum due to temperature or pH variations. On the other hand, adaptation of ppm can lead to a systematic overestimation of zero or small peak volumes, because the nearest local noise maximum is integrated as a peak. It is often a good compromise to adapt ppm, but restrict the adaptation to a very narrow range (e.g. Maximum change = 1/20 lw, Minimum relative distance = 0.98), and integrate the spectra in the order in which they were recorded without reading the reference peaklist in between.
In addition, to avoid misinterpretation of noise as peaks, the "volume error" entry in the peaklist should be checked automatically, and all peaks with values > 40...70 should be discarded.

Pick peaks and integrate a 3D spectrum

(Integration of hand-picked 3D peaklists is described below) Accuracy: In Spectra where a peak consists of only one pixel in one or two dimensions, and in spectra for which "sharpening" window functions were used, you get systematic volume errors of up to 30% for 3D.

This doesn't matter if you integrate all peaks with SPSCAN. But if you integrate some peaks "by hand" it my be worth to have a look, and apply a correction factor if neccessary.

Peaks with an obvious splitting can not (yet) been integrated with SPSCAN.

Integration of hand-picked 3D peaklists

Use the same procedure as described above, with the exception of the following parameter settings: In overlap regions it is neccessary that all peaks are picked, even if you are not interested in the volume of some of the peaks.

related commands: spm average_linewidth

Adjust calibration of several spectra

SPSCAN searches for peaks along strips, the position of which is taken from a peaklist obtained from another spectrum. It also takes the assignments from this list. Thus all spectra should be calibrated in a way that the resonances match as good as possible. In the case of small temperature or pH differences between the spectra it may even be an advantage to mis-calibrate one of the spectra slightly to get a better match for the average peaks.

The fine-adjustment of calibration is done with the following steps:

If you are just starting to pick a peaklist for this spectrum, then it is ususally a good idea to discard the current peaklist at that stage and repeat the peak-picking with the original list. Strips that could not adjust their position to the spectrum in the first run, should now have a better starting point. In the current (shifted) peaklist, they are now at a wrong position.

Change assignments in XEASY peaklists:

see also: Change assignment of one resonance below

The following situation may arise in XEASY: You have worked with two spectra in parallel, and inserted different atoms or assigned atoms from one residue to different fragments numbers. As a result, the peaklist from the two spectra cannot be used with the same proton list, and it is difficult to combine the information from the two peaklists. In XEASY this situation cannot be improved by mapping the fragments of both lists to the real sequence, since this does not change the assignment numbers in the peaklist, which remain incompatible.

SPSCAN offers a possibility to reassign the peaklists, i.e. to change the assignment numbers so that they correspond to a new proton list. You can also exchange fragment numbers without getting your proton lists mixed up. This allows, for example, to assign backbone, side chains, and aromatic rings independently and combine the information at the end.

The routine works in the following way: it uses the original XEASY proton list to resolve the fragment number and atom name. Then it searches in the new (target) proton list for the first entry that matches both values, and changes the assignment number to the number of this atom. If it does not find a matching atom and you have set "do atom mapping ?" = "y", it searches an atom mapping library for a valid conversion. Atoms for which no match is found become unassigned, and a message about the old status of the atom is printed out.

At the same time, you can change fragment numbers: set "do residue mapping ?" = "y".

Change assignment of one resonance

You want to change the assignment of a single resonance in a peaklist or in all peaklists of a project, e.g. all peaks that are currently assigned as LEU 105 HB3 should be assigned to LEU 105 HG.
The assignment numbers in the peaklist(s) are changed, the proton list is not immediately effected.
Procedure A: Make changes in a single peaklist
  1. 'project' 'peaklist manipulations'
  2. peaklist manipulations: 'load peaklist 1' (the peaklist must have a valid '#ASSIGN_MODE ...' line)
  3. peaklist manipulations: 'change assignment of a resonance'
  4. Enter the two assignments either as <atom name> <residue number> or as < assignment number>. To replace an intraresidual resonance you can omit <residue number> in the second line, if you have entered <atom name> <residue number> in the first line.
    Repeat this step until you have replaced all resonances.
  5. peaklist manipulations: 'save peaklist 1'
  6. peaklist manipulations: 'exit'
Procedure B: Make changes in all peaklists of the project
  1. 'project' 'edit'
  2. edit project: 'change assignment of a resonance'
  3. Enter the two assignments either as <atom name> <residue number> or as < assignment number>. To replace an intraresidual resonance you can omit <residue number> in the second line, if you have entered <atom name> <residue number> in the first line.
    Repeat this step until you have replaced all resonances.
  4. edit project: 'exit'
Exchange two assignments:
Change one of the assignments to an unused assignment number, e.g. 'HG 105' to '10599'; 'HB3 105' to 'HG 105'; '10599' to 'HB3 105'.
These procedures do not change the entries in the proton list. You have to make 'project' 'edit': 'check sequence and proton list'

Evaluate reduced dimensionality experiments and create a pseudospectrum

SPSCAN can extract strips from a spectrum and arrange them in such a way that the central peak is always in the same position. This allows the direct comparison of such strips with strips from normal experiments, and - to some extent - the use of XEASY strip correlation functions.
At the same time, processing in SPSCAN sets the relevant assignments and saves a lot of routine work. All routines are found under "reduced dim: HN_CO/CA". They are developed for COHNCA experiments and all explanations below refer to this type of experiment, but the routines can actually be used for any kind of 4D -> 3D reduced dimensionality spectra.

Before you start, you need a N-HN cosy list with the starting positions of the strips where you are searching for peaks. All entries in this list must be assigned, if they are not, use "assign"/"2 peak dim to inames" with residue SRD. In addition, you have to copy COHNCA-.scpa and COHNCA+.scpa into the current scpa_path and adapt the linewidth parameters (by reading the files in the parameter window, making the changes, and writing them out again).

The macro "COHNCA.spm" will do all of the steps described above, except adaptation of the parameters to your spectrum.

More detailed information is found under interface: HN_CO/CA

If there is more than one central peak per strip, several strips are created in the pseudo-spectrum. However, all peaks are shown in the first of these strips.

Referencing of the pseudo-spectra can be incorrect by up to +/- 1/4 pixel. Consider that lineshape comparison may be misleading for this reason.

Correlate spectra and dimensions of a spectrum

The following example shows a possibility to evaluate a 3D HC(C)H-COSY spectrum interactively. You learn how to use correlated crosshairs. With the same technique that is demonstrated here you can assign HN - Haliphatic NOE's from matching crosspeaks in a 15N-resolved and a 13C-resoved 3D-NOESY spectrum.

Let us assume that we have all HA-CA crosspeaks assigned. We want to find the other CH moieties of the sidechain. We start with a peak H1/C1 in a 2D overview spectrum "HC". In strip I of the 3D HC(C)H-COSY spectrum we find the diagonale peak H1/C1/H1 and one or two crosspeaks H1/C1/H2. To get C2 we have to find a matching crosspeak H2/C2/H1. This crosspeak must be visible on strip II. When we have found it, we display strip III to check whether the peaks are correctly centered and whether strip III shares other crosspeaks with strip I. From the crosspeak in strip II and III we can go back to the overview spectrum. The position of the strips in the spectrum is indicated below

HCCH drawing Now the steps to do interactively. In the 3D HC(C)H-COSY spectrum we have w1=H1, w2=C1, w3=H2. The spectrum is registered in the project as "HCCH", the 2D overview spectrum as "HC".

The display is now ready. It works as follows: The screenshot HCCH shows how the result should look like. The spectra used for this example are from P14a, and were kindly provided by Cesar Fernandez.

Some more hints:

alphabetic index / program documentation / database / internals / methods
Dr. Ralf W. Glaser
FSU Jena, Institut fuer Molekularbiologie
Winzerlaer Strasse 10
D-07745 Jena, Germany
Tel.: +49-3641-65-7573
Fax: +49-3641-65-7520

last changes: July 1998