Tutorial for Relaxation dispersion analysis cpmg fixed time recorded on varian as fid interleaved
Intro
This tutorial is based on the analysis of NMR data from the paper:
The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein.
Kaare Teilum, Flemming M Poulsen, Mikael Akke.
Proceedings of the National Academy of Sciences of the United States of America (2006).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1458987
The data is recorded as FID interleaved
Preparation
You want to make a working dir, with different folders
peak_lists
spectrometer_data
scripts
You can create the folders by
mkdir peak_lists spectrometer_data scripts
In the folder spectrometer_data should be the files: fid and procpar as the output from recording fid interleaved on Varian.
In the folder peak_lists should contain SPARKY list in SPARKY list format.
In the folder scripts we put scripts which help us processing the files.
Get the process helper scripts
cd scripts
# Change shell
tcsh
# Set array of scripts to download
set SCRIPTS="CPMG_1_sort_pseudo3D_initialize_files.sh CPMG_2_convert_and_process.sh CPMG_3_fft_all.sh convert_all.com fft_all.com sparky_add.sh stPeakList.pl NMRPipe_to_Sparky.sh"
# Download scripts
foreach SCRIPT ( ${SCRIPTS} )
curl https://raw.github.com/nmr-relax/relax_scripts/master/shell_scripts/$SCRIPT -o $SCRIPT
end
# Make them executable
chmod +x *.sh *.com *.pl
# Go back to previous directory
cd ..
Extract interleaved spectra, process to NMRPipe and do spectral processing
Extract interleaved and change format to NMRPipe
sort out the interleaved fid with the script CPMG_1_sort_pseudo3D_initialize_files.sh.
# Copy data
cp -r spectrometer_data spectrometer_data_processed
# sort_pseudo3D and initialize files
cd spectrometer_data_processed
CPMG_1_sort_pseudo3D_initialize_files.sh
Now we make a file to convert from binary format of Varian to NMRPipe.
- Now click, 'read parameters', check 'Rance-Kay'
- Remember to set Y-'Observe Freq MHz' to N15
- Click 'Save script' to make 'fid.com' file, and 'Quit', and run the next CPMG script
Now it is time to convert all the fid from varian format to NMRPipe with the script CPMG_2_convert_and_process.sh.
CPMG_2_convert_and_process.sh
Spectral processing
- Now we need to spectral process the spectra.
- Process one of the files normally and the next script will copy the processing script to the other folder.
- [m]->Right-Click Process 2D->Basic 2D
- Save->Execute->Done; then; RClick File->Select File->test.ft2->Read/draw->Done
- If your spectra look reversed (i.e. if your peaks do not seem to match your reference spectrum) it might be solved by changing to
- [m] '| nmrPipe -fn FT -neg \' to the script to the third lowest line.
- Save->Execute->Done. Then push [r] to refresh.
- Press [h], and find P0 and P1, and push [m], change parameters and update script
- The changes to '| nmrPipe -fn PS xxx \' should be the FIRST line (The proton dimension) with PS
- save/execute, push [r] (read) and the [e] (erase settings) to see result in NMRdraw
- And then run the next CPMG script
As suggested in the relax manaul, section 5.2.2 Spectral processing, the spectral processing script could look like: File: nmrproc.com
#!/bin/csh
nmrPipe -in test.fid \
| nmrPipe -fn SOL \
| nmrPipe -fn GM -g1 5 -g2 10 -c 0.5 \
| nmrPipe -fn ZF -auto -size 8000 \
| nmrPipe -fn FT -auto \
| nmrPipe -fn PS -p0 214.00 -p1 -21.00 -di -verb \
| nmrPipe -fn TP \
| nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
| nmrPipe -fn ZF -auto -size 8000 \
| nmrPipe -fn FT -neg \
| nmrPipe -fn PS -p0 0.00 -p1 0.00 -di -verb \
| nmrPipe -fn TP \
| nmrPipe -fn POLY -auto \
| nmrPipe -fn EXT -left -sw \
-ov -out test.ft2
Fourier transform all spectra
Now it is time to Fourier Transform all spectra with the script CPMG_3_fft_all.sh.
CPMG_3_fft_all.sh
Convert all *.ft2 files to ucsf format, so they can be opened in SPARKY
Done by the script NMRPipe_to_Sparky.sh
NMRPipe_to_Sparky.sh
Check the peak list matches
sparky 0.fid/test.ucsf