Scripts similar to these were originally created and compiled over several years, starting in 1991 at The Scripps Research Institute. The principal authors are Drs. Martin J. Stone, Johan Kordel, Mikael Akke, and Arthur G. Palmer. The scripts have been re-written to use the new program CurveFit and version 4 of the ModelFree program.
These scripts are free software; you can redistribute them and/or modify them under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or any later version.
These scripts are distributed in the hope that they will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.
The package should contain the following files:
cen1 is the F2 coordinate of the crosspeak
cen2 is the F1 coordinate of the crosspeak
asg1 is the F2 assignment of the crosspeak
asg2 is the F1 assignment of the crosspeak
int is the intensity of the crosspeak
vol is the volume of the crosspeak
Thus, the scripts contain the settings
NULL_NAME is assigned to be the string used if an assignment is not available for a residue (in either of the dimensions). For example, in FELIX, both the F1 and F2 assignments are identical for 1H-15N correlation spectra, so setting both assignments is not necessary.
The F1POS and F2POS fields are only needed for the checkpeaks_nmr script. This script is not essential for analyzing the relaxation data (see below).
Run the macro multiquant.mac. This macro will prompt you for a number of inputs:
The macro creates a temporary entity named 'peakmax'. If the macro crashes for some reason, you probably will have to delete this entity using the FELIX command
before reexecuting the macro.
The macro multiquant.mac has two modes set by the answer to the question 'use fixed peak locations (y/n)?'. In the first ('y'), the macro assumes that the assigned coordinates in the xpk:peaks entity represent appropriate peak locations. In the second ('n'), the macro searches for the maximum intensity within the region defined by the crosspeak widths in each dimension (scaled by the parameter 'fudge' defined in the macro). Normally, the second mode is preferable; however, for very weak peaks, multiquant.mac may find the peak maximum at a location that doesn't correspond to that where the stronger peaks have been found in a relaxation series. The script checkpeaks_nmr can be used to check for this problem (more below). A possible solution for the second mode for the spectra with strong intensities, and then the first mode for spectra with weak intensities.
Several of the scripts get their input by doing an `ls' and looking for all files present that conform (in some way) to the file name(s) provided on the command line. The draw-back with this is that you may need to think twice about how you name your files (and make sure that you keep rather clean directories), but the great benefit is that you never need to give the number of files, nor give a list of all of the files, as input.
Additional information on particular scripts is generally available in the header of that script. The appropriate command line syntax for the unix scripts will be printed if the script is executed without command line arguments. Remember that the input file field numbers will have to be changed in any script ending in 'nmr' if you processed the data using a program other than FELIX (see above).
in which rootname is the core filename, cut_off_w2 is the maximum number of points that you allow the locations of the peak maximum to differ in w2 between different spectra in the series, cut_off_w1 is the maximum number of points that you allow the locations of the peak maximum to differ in w1 between different spectra in the series, new_extension is for output files corresponding to each individual residue. One output file will be created for each record in the intensity files. The output filename will be given by the crosspeak name and output_extension (e.g. intensity) The crosspeak name is given by F2NAME_FIELD unless F2NAME_FIELD is equal to NULL_NAME, in which case the crosspeak name is given by F1NAME_FIELD. The script writes to standard output those instances where the peak maximum location for a certain crosspeak differs more than cut_off points from the average or median coordinates.
Copy the provided sample header file into the directory containing the intensity files and edit it as necessary. This file contains the commands to drive the CurveFit and xmgr programs. See the manual pages for these two programs for assistance in modifying the header file.
Run the nmr2curvefit script:
in which rootname is the core filename. masterfile and headerfile are the names of the master and header files created in the previous step. output_extension is the extension of the output file names. One output file will be created for each record in the intensity files. The output filename will be given by the crosspeak name (e.g. s74) and output_extension (e.g. t1) The crosspeak name is given by F2NAME_FIELD unless F2NAME_FIELD is equal to NULL_NAME, in which case the crosspeak name is given by F1NAME_FIELD. nmr2curvefit will take care of ambiguous crosspeak names; currently we have indicated ambiguity by incorporating a "/" or "?" in the crosspeak name in the (e.g. a12/g54, t24?, s45/, v34/k73?, e33/y99/w90, or any such combination). You can easily modify this to fit your own taste.
Run the curvefit_all script:
in which input_extension is the same as output_extension from the previous step andoutput_extension is the extension of the output file names for the CurveFit results. The optional parameters -grid, -jack, -xmgr -noerror are commands passed through to CurveFit. -print determines whether the xmgr plot should be printed to an output device (you should set the PRINTSTRING variable in the script appropriately). -display determines whether the xmgr plot should be displayed on the terminal.
Run the curvefit2table script:
in which extension is the output_extension from the previous step and X2_cutoff is the 1-alpha level for testing the quality of the fit (e.q. 0.95 for a 95% confidence level). The output contains the fitted rates for each crosspeak. A series of '*' are appended to each entry if the measured chi-square variable exceeds the critical value.
in which noe_spectrum and no_noe_spectrum are intensity files produced using FELIX or other processing program as described above. Output_file is contains the crosspeak name and the noe, i.e. the ratio of the peak intensity in noe_spectrum and no_noe_spectrum [i.e. intensity(noe)/intensity(no_noe)] as calculated from both the peak heights and volumes. If uncertainty estimates are provided for the input spectra, then uncertainties will be calculated for the NOE as well.
If multiple NOE data sets have been acquired, then average values can be calculated as either conventional mean values or as weighted mean values using the scripts:
in which noe_file_N are the output from the noecalc_nmr script. Weighted averages only can be calculated if uncertainties were obtained in the output from the noecalc_nmr script.
in which r1table and r2table were produced using the curvefit2table script and noetable were produced from noecalc_nmr, noe_average, or noe_weighted_average scripts. height/volume parameter determines whether NOE values based on peak heights or peak volumes will be used.
Do the above for each static magnetic field for which you have data. You will then have one table of R1, R2, NOE data for each static field. To create the ModelFree data file, run the script:
in which field_N is the 1H Larmor frequency in MHz for the Nth field and rates_table_N is the output from the make_ratestable script for the Nth field.
For further notes and help, please first take a look in the header of the script in question, or look at the man pages of the ModelFree and CurveFit programs.