Processing 2D NMR Spectra

Procedures for processing varian 2D NMR spectra from raw FID to ucsf format sparky input files. 


red text - what you are doing

blue text - commands you enter by typing them in

green text - menu buttons you click on 

bold items - files

Processing 3D Data

Processing T1/T2 Relaxation Data

Processing Interleaved RDC Data

Processing Interleaved NOE Relaxation Data

Processing Transverse Dipole Chemical Shift Anisotropy (CSA) Cross-Correlation Experiments (ηxy)

link to another site that discusses 2 and 3D processing

With a Unix shell open to the directory containing the data.fid file:

unzip file if necessary,

type 'gunzip -9 -v data.fid.gz', enter

convert varian data into nmrPipe format,

type 'varian' on command line, enter. Click 'Read Parameters'. Check all yellow highlighted items. Click  'Save script' (saves Click 'Execute script' (creates test.fid). Click 'Quit'.

open nmrDraw, visually inspect data, and get phasing information, 

type 'nmrDraw' on command line.  A new window will open.  Use right mouse button to select items from the menu at the top.  Right click on  'Select File'.  Double click on the test.fid file you made in the previous step. Click 'Done'. Right click on 'Mouse'.  Select '1D  Horizontal'. Look at 1st FID (enter y=1).  Right click 'Proc', then 'NMRPipe Commands'. Right click 'Functions', then 'Solvent Filter'. click 'Execute'. (suppresses the water signal, ex. SOL -fl 16 -fs 2 or POLY -time).  Right click 'Cosine Bell'. click 'Execute'. (Enter value in the range of 0-0.5 after the -off flag; smaller number gives sharper lines but lower resolution, ex. SP -off 0.5 -end 1.0 -pow 1).  Right click 'Zero Fill'. Replace -auto flag with -size 4096. Click 'Execute'. (adds zeros to the end of the FID which improves resolution for purely mathematical reasons, want the final result to be a power of 2, ex. ZF -size 4096).  Right click 'Fourier Transform'. click 'Execute'. Use the automatic version.  You are now ready to obtain the phasing information (values of P0 and P1 which give a continous baseline).  Click Phasing 'On'.  Slide the P0 slide ruler until the baseline before and after the signal are as continous as possible. P0 is a rough phasing, while P1 provides finer control.  Make note of the P0 and P1 phasing numbers.  Right click 'Baseline correction'.  click 'Execute'.  Use the automatic version (ex. POLY -auto -ord 1).  Examine the x-axis values.  Often, the x-axis is in points.  In this example, the points on the x-axis ranged from 0-1025 - which represents all of the data plus the zero filling up to 4096.  Therefore, the 1025 points represent values from 0-4096.  So if you want to extract the signal between 343 and 458 points on the x-axis, you will be extracting values (350 x 4096)/1025 = 1370 and (450 x 4096)/1025 = 1830.  These are the numbers you will place into the example macro shown below.

Leave nmrDraw open while opening another shell - change to the directory containing your fid.  Create the following file in this directory -  The following is a generic version of this script which contains all of the steps previously described in the above nmrDraw section.

nmrPipe -in test.fid                                                         \
| nmrPipe  -fn POLY -time                                             \
| nmrPipe  -fn SP -off 0.5 -end 1.00 -pow 1 -c 0.5 \
| nmrPipe  -fn ZF -size 4096                                         \
| nmrPipe  -fn FT -auto                                                    \
| nmrPipe  -fn PS -p0 -72.0 -p1 0.00 -di -verb         \
| nmrPipe  -fn POLY -auto -ord 1                                  \
| nmrPipe  -fn EXT -x1 1370 -xn 1830 -sw                 \
| nmrPipe  -ov -out test.ft2

Replace all of the pink text with the values that you chose/determined during your nmrDraw processing of the data. 

Once you have created, make it an executable file (Linux) by using the command chmod a+x pipe_d1.comThen run it by typing onto the command line.  This will create the file test.ft2, which has been processed in the proton dimension only.

The following generic script is for processing the indirect dimension of 2D data. 

nmrPipe -in test.ft2                                                         \
| nmrPipe  -fn TP                                                                 \
| nmrPipe  -fn SP -off 0.5 -end 0.98 -pow 1 -c 1.0    \
| nmrPipe  -fn ZF -size 1024                                              \
| nmrPipe  -fn FT -auto                                                      \
| nmrPipe  -fn PS -p0 -90.0 -p1 180.0 -di -verb        \
   -inPlace -out test.ft2

The TP function transposes the axis that is being processed from the direct to the indirect.  There are no solvent filter, baseline correction, or extraction functions for the indirect dimension.  Generally the p0 and p1 phasing values for the indirect dimension are fine at -90 and 180, but you may want to check this in nmrDraw (by analyzing the test.ft2 file created from in the same way you did for the direct dimension.

Create the file in the directory containing test.ft2.  Run the file by making it executable and typing it onto the command line as described above for

Change the resulting test.ft2 file to Sparky format, if desired, by using the following command: pipe2ucsf test.ft2 test.ucsf.  Or change test.ft2 into nmrView format using the following command: nmrPipe -in test.ft2 | pipe2xyz -nv -ov -out test.nv