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NOTE FOR INSTRUCTORS (Georgia State and Georgia Tech):
 

Nothing new is needed for this computer lab.

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GOALS:

1) Introduction to the visualization package NMRDraw

 2) Complete processing of a 2D 1H,15N HSQC
 
 

INTRODUCTION TO NMRDRAW:
 

The NMRDraw program provides facilities for inspecting raw and processed data via 1D and 2D slices or projections from all dimensions.  NMRDraw also allows real-time display and interactive phasing and processing of an arbitrary number of 1D slices selected from any dimension of the spectrum and displayed simultaneously.  It is very useful when used in combination with nmrPipe.
 

INSPECTION OF RAW AND TRANSFORMED DATA:

Last week, we transformed a 2D 1H,15N HSQC using the two UNIX shell scripts fid.com (for converting the data) and ft2.com (for processing the data).  To get back to these data type:

%cd bcmb8190/nmrpipe/hsqc.fid

This week, we will inspect the raw and processed data and decide if any additional processing is required, for instance re-phasing or baseline correction.  This step will be performed using the interactive graphical interface nmrDraw.  To start nmrDraw simply type:

%nmrDraw

A window should appear with menu options on the top panel display.  This menu is accessible by using the right mouse button (RMB) or by typing the letter that correspond to the desired function while the cursor is in the spectra display area.

A) Lets first open our raw data set (type "s" or under File, Select File, click on fid.DAT, click on Real/Display, and when the display is completed click on "done")

B) Lets observe 1D vertical and horizontal  slices to see the FID in both dimensions (type "v" or "h" or select  Mouse, 1D Horizontal or 1D Vertical).  Move the 1D slice using the left mouse button (LMB).  You can change the intensity of these slices by first placing the cursor on the right side of the spectrum, click and drag middle mouse button (MMB) up or down.  To get out of the vertical slice or horizontal slice modes, select Mouse, Exit Mode or simply type "e". [For more advanced users, try to select the first 1D horizontal slice and process it interactively.]

C) Lets now open our processed data (hsqc.DAT) as done for fid.DAT and observe 1D vertical and horizaontal slices.

D) Lets zoom on the data.  Select Mouse, 2D Zoom or type "z".  Select the region of interest by clicking and dragging the edges of the box using the RMB. For now make sure that you include all the signals (not the water).  To get out of the zooming mode type "e". Lets change the level of the data by adjusting the + and - in the top panel display.

E) You notice that this spectrum is not well phased in the F2 dimension.  Lets try to phase it.  For this purpose it is good to displayed various 1D horizontal slices at the same time.  I usually select two slices, one containing the most downfield signals and one containing the most upfield signals.  You will see how useful this is to adjust the first order phase correction.  Lets select one of the two slices by placing the horizontal slice on the proper row (click and drag with LMB) and type "a".  This will "freeze" the horizontal display on this slice.  You can now choose another slice using the same procedure. Adjust p0 and p1 in the top panel display until you get the right phasing.  Record the values of p0 and p1.
 

REPROCESSING THE DATA:

A) Rephase - Adjust the phasing command in ft2.com by adding the new values of phasing that you found and rerun ft2.com:

| nmrPipe  -fn PS -p0 -3.0 -p1 0.0  -di   \

Visualize the results with nmrDraw and readjust phasing if still not satisfactory.

B) Remove right part of spectra -Rerun ft2.com after adding the following nmrPipe command at the end:

| nmrPipe  -fn EXT -left -sw     \

Visualize the results with nmrDraw and print the data (Select File, Hard Copy Plot (Plot Command lp) or type "P").
 

CONVERTING NMRPIPE TO NMRVIEW FORMAT

In the next two computer labs, we will use NMRView, which is a powerful program for data analysis. We need convert the 2D data processed using nmrPipe to the NMRView format. This is simply done with the shell script "nmrview.com". Lets view this file and then run it:

%vi nmrview.com
%nmrview.com
%ls  (A new file should be created called hsqc.nv.)
 
 

LAB PROBLEM SET #8:
 

Here are a few exercises to help you get familiar with this type of data and review of few basic principles that we have seen (or will see) in class.  For your information, the sequence of this peptide is:

G  S  M  D  A  Q  T  R  R  R  E  R  R  A  E  K  Q  A  Q  W  K  A  A  N

A) Using the Mouse, 2D location or type "l" get chemical shift values of the side chain NH2 signals for this 24 amino acid peptide (from asparagine (N) and glutamine (Q) residues).

B) Note that NH2 signal comes as a pair of peaks with the same 15N frequency, but different 1H frequencies.  Also characteristic of these signals is the deuterium isotope effect, which shift a fraction of the 15N signal by about 0.3 ppm.  Since there is about 10% of D2O in this sample, there are various populations of NH2, NHD, and ND2.  Can you estimate the population of these amino groups ?  Can you estimate the relative ratio of the 15N signals originating from NH2, and NHD ?

C)  The 5 signals observed between 107 ppm and 111 ppm in the 15N dimension originates from Ne-He correlations in the arginine side chains.  These signals are aliased in this spectrum.  Calculate their real chemical shifts knowing that the Ne are usually found around 85 ppm.

D)  The 2 signals observed around 128 ppm in the 15N dimension originates from the ammonium group of lysine side chains.  These signals are also aliased in this spectrum.  Calculate their real chemical shifts knowing that they usually resonate around 33 ppm.

E)  In A) B) and C) and D) we have analyzed most side chain signals observed in this spectrum.  There is also a tryptophan indole signal at 129 ppm.  All the other intense signals comes from the backbone amides.  How many amide signals do you expect? Can you count them?