Tutorial for Relaxation dispersion analysis r1rho fixed time recorded on varian as sequential spectra
Contents
Intro
This tutorial is not complete.
This tutorial is based on the analysis of R1rho data, analysed in a master thesis.
The spectra is not recorded interleaved, but as a series of spectra with experimental changes.
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 peak_lists should contain SPARKY list in SPARKY list format.
In the folder scripts we put scripts which help us processing the files.
In the folder spectrometer_data should be a directory containing directories with all experiments where each directory contain files: fid and procpar as the output from recording on Varian.
Establish file-overview
Make file with paths to fid files
We make a file list of filepaths to fid files.
ls -v -d -1 */fid > fid_files.ls
cat fid_files.ls
Do something in each directory
With the fid_files.ls, we can do something in each directory.
For example do a list files in each direcory.
set FIDS=`cat fid_files.ls`
foreach I (`seq 1 ${#FIDS}`)
set FID=${FIDS[$I]}; set DIRN=`dirname $FID`
cd $DIRN
ls
cd ..
end
Extract the spectra settings from Varian procpar file
Now we want to make a settings file we can read in relax.
set FIDS=`cat fid_files.ls`
set OUT=$PWD/exp_parameters.txt
echo "# DIRN I deltadof2 dpwr2slock ncyc trim ss sfrq apod_rmsd" > $OUT
foreach I (`seq 1 ${#FIDS}`)
set FID=${FIDS[$I]}; set DIRN=`dirname $FID`
cd $DIRN
set deltadof2=`awk '/^deltadof2 /{f=1;next}f{print $2;exit}' procpar`
set dpwr2slock=`awk '/^dpwr2slock /{f=1;next}f{print $2;exit}' procpar`
set ncyc=`awk '/^ncyc /{f=1;next}f{print $2;exit}' procpar`
set trim=`awk '/^trim /{f=1;next}f{print $2;exit}' procpar`
set ss=`awk '/^ss /{f=1;next}f{print $2;exit}' procpar`
set sfrq=`awk '/^sfrq /{f=1;next}f{print $2;exit}' procpar`
set apodrmsd=`showApod test.ft2 | grep "REMARK Automated Noise Std Dev in Processed Data:" | awk '{print $9}'`
echo "$DIRN $I $deltadof2 $dpwr2slock $ncyc $trim $ss $sfrq $apodrmsd" >> $OUT
cd ..
end
cat $OUT
You can check, if you have repetitions of experiments, by sorting the parameters, and see if they are dublicated.
We do this, by numerical sort columns 3,4 and 5 with the values for "deltadof2, dpwr2slock, ncyc".
sort -b -k 3,3n -k 4,4n -k 5,5n exp_parameters.txt | awk '{print $3, $4, $5}'
sort -b -k 3,3n -k 4,4n -k 5,5n exp_parameters.txt > exp_parameters_sort.txt
Spectral process files
Copy data
We first copy the data
# Copy data
cp -r spectrometer_data spectrometer_data_processed
cd spectrometer_data_processed
Change format to NMRPipe
set CWD=$PWD
set DIRS=`cat fid_files.ls | sed 's/\/fid//g'`
cd ${DIRS[1]}
varian
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
- Remove sleep 5 from the script.
- Click 'Save script' to make fid.com file, and 'Quit', and run the script.
Spectral processing
This step can be done by following wiki page Spectral_processing.
Start nmrDraw by command
nmrDraw
Convert and spectral processing all
Now we want to convert all spectra.
You should have a fid.com and nmrproc.com in the first FID folder.
We now copy these script into all of the experimental folders, and execute them.
cd $CWD
set FIDS=`cat fid_files.ls`
set DIRN1=`dirname $PWD/${FIDS[1]}`
foreach I (`seq 2 ${#FIDS}`)
set FID=${FIDS[$I]}; set DIRN=`dirname $FID`
cd $DIRN
echo $DIRN
cp -f $DIRN1/fid.com .
cp -f $DIRN1/nmrproc.com .
./fid.com
./nmrproc.com
cd ..
end
Convert NMRPipe to Sparky
Next we also want to convert them to SPARKY format.
set FTS=`ls -v -d -1 */*.ft2`
foreach FT ($FTS)
set DNAME=`dirname $FT`
set BNAME=`basename $FT`
set FNAME=`echo $BNAME | cut -d'.' -f1`
echo $FT $DNAME $BNAME $FNAME
pipe2ucsf $FT ${DNAME}/${FNAME}.ucsf
end
Working with peaks
Check the peak list matches
Check that your peak list matches your spectrum.
Read the section in Check the peak list matches.
set DIRS=`cat fid_files.ls | sed 's/\/fid//g'`
sparky ${DIRS[1]}/test.ucsf
The final peak list is expected to be in:
/peak_lists/peaks_corr_final.list
And have been saved by SPARKY, so it is in this format SPARKY_list.
Check for peak movement
Your should check, that the peaks do not move at the different experiments. Try opening some random spectra, and overlay them in SPARKY.
Read the section in Check for peak movement.
sparky ${DIRS[1]}/test.ucsf ${DIRS[10]}/test.ucsf ${DIRS[25]}/test.ucsf ${DIRS[50]}/test.ucsf
Measuring peak heights
We will use the program NMRPipe seriesTab to measure the intensities.
seriesTab needs a input file, where the ppm values from a SPARKY list has been converted to spectral points.
The spectral points value depends on the spectral processing parameters.
Generate spectral point file
Create a file with spectral point information with script stPeakList.pl .
stPeakList.pl ${DIRS[1]}/test.ft2 ../peak_lists/peaks_corr_final.list > peaks_list.tab
cat peaks_list.tab
Make a file name of .ft2 fil
echo "test.ft2" > ft2_file.ls
Measure the height or sum in a spectral point box
mkdir peak_lists
foreach line ("`tail -n+2 exp_parameters.txt`")
set argv=( $line )
set DIRN=$1
set I=$2
set deltadof2=$3
set dpwr2slock=$4
set ncyc=$5
set trim=$6
set ss=$7
set sfrq=$8
echo $I
set FNAME=${I}_${deltadof2}_${dpwr2slock}_${ncyc}
cd $DIRN
seriesTab -in ../peaks_list.tab -out ${FNAME}_max_standard.ser -list ../ft2_file.ls -max
seriesTab -in ../peaks_list.tab -out ${FNAME}_max_dx1_dy1.ser -list ../ft2_file.ls -max -dx 1 -dy 1
seriesTab -in ../peaks_list.tab -out ${FNAME}_sum_dx1_dy1.ser -list ../ft2_file.ls -sum -dx 1 -dy 1
cp ${FNAME}_max_standard.ser ../peak_lists
cd ..
end
Analyse in relax
Preparation
Prepare directory for relax run
Then we make a directory ready for relax
mkdir ../relax
cp exp_parameters.txt ../relax
cp exp_parameters_sort.txt ../relax
cp -r peak_lists* ../relax
cp peaks_list.tab ../relax
cd ../relax
See unique parameters
tail -n +2 exp_parameters.txt | awk '{print $3}' | sort -k1,1n | uniq
tail -n +2 exp_parameters.txt | awk '{print $4}' | sort -k1,1n | uniq
tail -n +2 exp_parameters.txt | awk '{print $5}' | sort -k1,1n | uniq
tail -n +2 exp_parameters.txt | awk '{print $6}' | sort -k1,1n | uniq
tail -n +2 exp_parameters.txt | awk '{print $7}' | sort -k1,1n | uniq
tail -n +2 exp_parameters.txt | awk '{print $8}' | sort -k1,1n | uniq
Scripts
1_setup_r1rho.py
This a script file to be able to call the setup.
file: 1_setup_r1rho.py.
# Python module imports.
from os import getcwd, sep
# relax module imports.
from data_store import Relax_data_store; ds = Relax_data_store()
#########################################
#### Setup
# The pipe names.
if not (hasattr(ds, 'pipe_name') and hasattr(ds, 'pipe_bundle') and hasattr(ds, 'pipe_type')):
# Set pipe name, bundle and type.
ds.pipe_name = 'base pipe'
ds.pipe_bundle = 'relax_disp'
ds.pipe_type = 'relax_disp'
# The data path
if not hasattr(ds, 'data_path'):
ds.data_path = getcwd()
#########################################
### Start setup
# Create the data pipe.
pipe.create(pipe_name=ds.pipe_name, bundle=ds.pipe_bundle, pipe_type=ds.pipe_type)
# Read the spins.
spectrum.read_spins(file='1_0_46_0_max_standard.ser', dir=ds.data_path+sep+'peak_lists')
# Name the isotope for field strength scaling.
spin.isotope(isotope='15N')
# Load the experiments settings file.
expfile = open(ds.data_path+sep+'exp_parameters_sort.txt', 'r')
expfileslines = expfile.readlines()
expfile.close()
# In MHz
yOBS = 81.050
# In ppm
yCAR = 118.078
centerPPM_N15 = yCAR
## Read the chemical shift data.
chemical_shift.read(file='1_0_46_0_max_standard.ser', dir=ds.data_path+sep+'peak_lists')
## The lock power to field, has been found in an calibration experiment.
spin_lock_field_strengths_Hz = {'35': 431.0, '39': 651.2, '41': 800.5, '43': 984.0, '46': 1341.11, '48': 1648.5}
## Apply spectra settings.
for i in range(len(expfileslines)):
line = expfileslines[i]
if line[0] == "#":
continue
else:
# DIRN I deltadof2 dpwr2slock ncyc trim ss sfrq
DIRN = line.split()[0]
I = int(line.split()[1])
deltadof2 = line.split()[2]
dpwr2slock = line.split()[3]
ncyc = int(line.split()[4])
trim = float(line.split()[5])
ss = int(line.split()[6])
set_sfrq = float(line.split()[7])
apod_rmsd = float(line.split()[8])
spin_lock_field_strength = spin_lock_field_strengths_Hz[dpwr2slock]
# Calculate spin_lock time
time_sl = 2*ncyc*trim
# Define file name for peak list.
FNAME = "%s_%s_%s_%s_max_standard.ser"%(I, deltadof2, dpwr2slock, ncyc)
sp_id = "%s_%s_%s_%s"%(I, deltadof2, dpwr2slock, ncyc)
# Load the peak intensities.
spectrum.read_intensities(file=FNAME, dir=ds.data_path+sep+'peak_lists', spectrum_id=sp_id, int_method='height')
# Set the peak intensity errors, as defined as the baseplane RMSD.
spectrum.baseplane_rmsd(error=apod_rmsd, spectrum_id=sp_id)
# Set the relaxation dispersion experiment type.
relax_disp.exp_type(spectrum_id=sp_id, exp_type='R1rho')
# Set The spin-lock field strength, nu1, in Hz
relax_disp.spin_lock_field(spectrum_id=sp_id, field=spin_lock_field_strength)
# Calculating the spin-lock offset in ppm, from offsets values provided in Hz.
frq_N15_Hz = yOBS * 1E6
offset_ppm_N15 = float(deltadof2) / frq_N15_Hz * 1E6
omega_rf_ppm = centerPPM_N15 + offset_ppm_N15
# Set The spin-lock offset, omega_rf, in ppm.
relax_disp.spin_lock_offset(spectrum_id=sp_id, offset=omega_rf_ppm)
# Set the relaxation times (in s).
relax_disp.relax_time(spectrum_id=sp_id, time=time_sl)
# Set the spectrometer frequency.
spectrometer.frequency(id=sp_id, frq=set_sfrq, units='MHz')
# Read the R1 data
# We do not read the R1 data, but rather with R1.
# relax_data.read(ri_id='R1', ri_type='R1', frq=cdp.spectrometer_frq_list[0], file='R1_fitted_values.txt', dir=data_path, mol_name_col=1, res_num_col=2, res_name_col=3, spin_num_col=4, spin_name_col=5, data_col=6, error_col=7)
