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NOTE FOR INSTRUCTORS (Georgia State and Georgia Tech):
 

See below (Install NMRview and necessary files for the tutorial).

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GOALS:

1) Introduction to the NMR analysis package NMRView.

2) Introduction to sequential assignment principles using 3D HNCO, 3D HNCACB and 3D CBCACONNH experiments.
 
 

INTRODUCTION TO NMRView:
 

    The NMRView is a software developed by Bruce A. Johnson at Merck Research Laboratories for vizualisation and analysis of multidimensional NMR data.  For this class we will learn the basics of NMRView and next week, we will use it for sequential assignment of a 24-amino acid peptide using triple resonance experiments.
 

INSTALL NMRView AND NECESSARY FILES FOR THE TUTORIAL
 

1.  ftp or copy to mgl computers to get the nmrview files:

FTP (for people at Georgia Tech and Georgia State)

The .tar file is called nmrview.tar and is found in the directory /21/users/pascale/bcmb8190_00. To untar:
%tar -xvfh nmrview.tar

COPY (For People at UGA)

%cd bcmb8190
%cp -r  /21/users/pascale/bcmb8190_00/nmrview .

this will give you a directory called nmrview with all the necessary files

2.  Adapt my files to your computer:

You need to change a few lines in the file hsqc.tcl.  Open the file hsqc.tcl and replace all occurence of the directory /21/users/pascale/bcmb8190/nmrview/ or  /21/users/pascale/bcmb8190_00/nmrview/  to your nmrview directory (the directory where you have the .nv files (pl181t.nv, pl198t.nv, pl185t.nv, pl206t.nv)).  Open the file MYDatabase5.str and change directory /21/users/pascale/bcmb8190/nmrview/ to your nmrview directory.  Also include the following lines in your .cshrc file:

 setenv NMRVIEW4HOME /usr/chem/nmrview
 setenv TK_LIBRARY /usr/chem/nmrview/tk8.0
 setenv TCL_LIBRARY /usr/chem/nmrview/tcl8.0

(Directories may be different at Georgia Tech and Georgia State)
Source your .cshrc files by typing:
%source .cshrc

You should be ready to start.
 

START THE NMRVIEW EXECUTABLE

%nmrview (or the name or your executable)

Once you started NMRView you should get
 1) an nmrview menu window
 2) an NMRView Console window
 3) an info window that'll disapear after a few seconds.
 

GET HELP AND OTHER INFO

From the nmrview menu window go to "help" and then "User's manual (Internal Viewer)"

Go to "History and Acknowledgments" to get a brief overview of the origin of NMRView.

In this User's manual, you can also find a lot of very useful and easily accessible information about the software.
 

GETTING STARTED: 2D ¹H-¹⁵N HSQC

We take the HSQC of an ¹⁵N-labeled N-peptide / boxB RNA complex as an example.  This is the same HSQC that you transformed using NMRPipe and analyzed using NMRDraw.  For more info on this RNA-peptide complex:  Legault et al. Cell 1998, vol.93, 289-299.
 

The sequence of the N-peptide is:

-2 -1   1     2    3   4    5   6    7   8    9   10 11  12 13  14  15  16  17 18   19  20  21  22
 g   s   M   D   A   Q   T   R   R   R   E   R   R   A   E   K   Q   A   Q   W   K   A   A   N
 

1.  From the nmrview menu go to "Dataset" and then "open dataset". Open the file
pl181t.nv from you nmrview directory.

2.  Now select "add" under the "Windows" menu. Give hsqc (lower case letters)
for the Window Name and hit the "Create" button.

3.  Direct the mouse to this hsqc window and click the right button.  You should
get a menu with various options. Click on "Attributes".  You should get a new window called ".specAttr:.hsqc.0". Go under file, select "dataset".  You will get a new window which should show all the dataset that you have open so far;  so you should only see now the file pl181t.nv. Highlight this filename and click on the "Add" button. Then close this window by clicking on the "close" button.

4.  Move the Attributes window away from the hsqc window.  Increase the size of the hsqc window and click on the "Draw" button.
You should see the spectrum being drawn in the hsqc window.

5.  Play with the attribute window to change the display of the hsqc. (Note:  Always click on Draw after changing any settings)

Lvl: Level: changes the peak level. Try Lvl at 0.04
CLM: Contour Level Multiplier: adjust the spaces between the different number of contours drawn.  Try 1.0 than 2.0, than back to 1.2.
NLvls: Number of Levels: adjust the number of levels draw Try 1, 2, back to 20.

You can adjust the spectral window by directly changing the chemical shift bounds in the Attribute window. Alternatively, you can place the two cursors in the window (with the left and middle mouse button) to define a subregion of the spectrum that you would like to expand. Once you have defined this region, click on the right mouse button, then "View", then "Expand".
If you want to get back to the orginal display, click on the right mouse button again, and "View" and "Previous" or "Full".

Click on the right mouse button, and select "Slice", click on x to get an x slice. You can adjust the display of slices with the "Scale", "XOff", and "YOff" in the Attributes window. Everytime you make an adjustement, you have to hit the "Draw" button.
 

6.  Shortcuts

With cursor in the hsqc window, click on the right mouse button and go to "misc" "KeyBinding". Type the following in the
appropriate spaces:

e: nv_win expand;     f: nv_win full;     p: nv_win previous;     d: nv_win draw

and close the window. Now if you want to draw your spectrum, you simply need to type "d" when the mouse is in the hsqc window. (similarly for other commands that we defined). It is much faster and convenient.

7.  Peak picking

Expand to a small region of the spectrum and click on the right mouse button, then "Peak", then "Pick". Give "hsqc" as the List Name, then select "Window", "New", "2", and click on Pick. You can assign, delete, or modify the peak lists as follows:

From the nmrview menu select "Analysis" and "Peaks". Click on list.hsqc and you should get the hsqc list. You can assign peaks by replacing the first "?" by the name of the atom (30.HN and 30.N for example). You can delete a peak by first going to the appropriate peak.  For example, if you want to delete peak "5", type "5" and return in the top entry bar and then return. Hit the dead head, which should then take a red color. If you draw the spectrum at this point, the box around this peak will not appear. To completely remove this peak from the list, go under "Edit" and "Compress".
 

8.  tcl scripts for automated tasks.

Now lets exit the program. Type exit in the Console window.

Restart nmrview by typing:

nmrview -- -b MYDatabase5.str

In the Console window type:
>source hsqc.tcl
>hsqc

Wow! All the work we did can be repeated with only a few command lines. Go see the macro hsqc.tcl to try to understand how to program in tcl language. You can also use the shortcuts that we defined!  The peak table has been saved (database file MYDatabase5.str); see Analysis/Peak from the Main Menu.
 

3D DATASET FOR PROTEIN SEQUENTIAL ASSIGNMENTS
 

1. Exit the program and restart by typing:

nmrview -- -b MYDatabase5.str

2.  In the Console window type:

>source hsqc.tcl
>seqass
>source jump.tcl
 

You will get 4 windows, each one containing a single dataset: The same hsqc experiment, and hnco, a hncacb, and a cbcaconnh. I have already created a peak list for each of these experiments.

3.  Look at the data in the 3D experiments just to have an idea.  You can use the up and down arrows to go up and down in the 3D planes.  Also the "Attributes" window is very useful to follow the ¹⁵N frequency of the plane that is currently displayed.  Make sure that you adjust the Level (Lvl) of the 3D spectra to see the weakest signals!
 
 

LAB PROBLEM SET #9:
 

1.  Threonine 7 has amide chemical shifts of 8.38 ppm in ¹H and 118.8 ppm in ¹⁵N.  Locate the crosspeak in the 3D HNCO which corresponds to these ¹H and ¹⁵N frequencies.  What is the carbonyl frequency (in ppm) associated with these ¹H  and ¹⁵N frequencies ?  This carbonyl frequency belong to which amino acid in the N peptide ?

2.  Locate the crosspeak in the CBCACONNH which corresponds to these ¹H and ¹⁵N frequencies.   What are the aliphatic carbon frequencies (in ppm) associated with these ¹H  and ¹⁵N frequencies ?  This aliphatic carbon frequencies belong to which group (alpha, beta, gamma, etc.) and which amino acid in the N peptide ?

3.  Locate the crosspeak in the HNCACB which corresponds to these ¹H and ¹⁵N frequencies.   What are the aliphatic carbon frequencies (in ppm) associated with these ¹H  and ¹⁵N frequencies ?  These aliphatic carbon frequencies belong to which group (alpha, beta, gamma, etc.) and to which amino acids in the N peptide ?

4.  On the basis of what you did in question 1, 2, 3,  can you now find the ¹H and ¹⁵N chemical shift of Arginine 8?