Using Model

When you are verifying the results of structure generation and refinement based on NMR spectra it is very important to visually inspect and compare the experimental spectra with the back-calculated spectra, together with the molecular model. These tasks can be carried out in the Model module and are presented in the following lesson.

In Lesson 1: Back-calculating NOE spectra, you will learn:

Topics covered in this lesson include:


Lesson 1: Back-calculating NOE spectra

This lesson demonstrates two uses of the Model module.

1.   Setting up for the lesson

You should keep in mind that the Model module requires that you have a database file containing picked, integrated, and assigned cross peaks.

The following files from the $BIOSYM/tutorial/felix directory are required for this lesson. Please copy these files to your current directory:

zb.mat
model.dba
znrddg.car
znrddg.mdf

2.   Starting FELIX and opening the model database

In your working directory, enter felix at the system prompt to start the program. If you get the RESTORE LAST SESSION dialog box, select CANCEL.

Select File/Open, and set File Type to DBA. In the Files list specify model.dba and select OK to build a new database.

If you are continuing from another lesson, select the File/Close menu item and set the File Type option to DBA, then select OK to close the current database.

Now select the File/Open menu item, select model.dba from the Files list box, and assure that the File Type is set to DBA (*.dba). Select OK.

This database file contains all the information you need in this tutorial.

3.   Open the experimental matrix

Select the File/Open item from the menubar or the Open File icon from the iconbar. Set File Type to Matrix and select zb.mat from the Files list box. Select OK.

If the automatically set threshold is too low (for example, less than 0.1) use the Increase Threshold icon to have less noise displayed.

4.   Open the structure module

Select the Model/Setup/Connect menu item. Set these parameter values:

Draw Molecule in FELIX
Experimental Frame 1
Peak Table xpk:buildup
Volume Table vol:buildup
Theoretical Frame 2
Molecule Frame 3
Arrange Frames on

Select OK.

If you are running the SUN version of FELIX, you do not have the option of using the Insight window to display the molecule, therefore the Use Insight Window control is not present.

The frame layout changes automatically. In the right frame you see the molecule, and in the left frame the experimental NOESY spectrum of this compound.

5.   Adjust the display

Activate the first frame by clicking in it. Now select the Preference/Plot Parameters menu item to change the display of the spectrum. Change the Contour Threshold to 0.1 and the Color Scheme to Fire Ramp and click OK.

Select the View/Draw Peaks menu item.

Multiple cross peaks appear.

Select the View/Limits/Set Limits menu item and use the cross hair to draw a box around a region of the spectrum.

This zooms in on a smaller region of the spectrum. Try to select a region between 8 and 5 ppm.

6.   Back-calculate the theoretical NOESY spectrum

Select the Model/Matrix Doubling menu item.

In Model you may do a back-calculation of NOESY spectra or read in an already back-calculated NOESY spectrum from the Insight IRMA module.

The back-calculation algorithm in FELIX is based on matrix doubling and is faster than the IRMA process.

Set these parameter values:

New Crosspeak Table bck:xpk
New Volume Table bck:vol
Mixing Time (sec) 0.45
Corr. Time (nsec) 1.5
Z-leakage (1/sec) 3
Cutoff Radius (A) 7
Minimum Intensity 0.0050
Spect Freq (MHz) 600
Frame to Draw Theoretical 3
Molecule Use Current

Select OK.

You see a message in the text window:

     Back-calculating spectrum

and after a short while:

     Done back-calculating

Next you choose a scaling peak. This peak should have the same intensity in both the experimental and back-calculated spectra.

In the next control panel, set the Method to Enter and enter a Peak Name D1 of D1 1:ASP-_13:HB1 and a Peak Name D2 of 1:ASP-_13:HB2. Select OK.

You select this peak to have the same intensities in the two spectra. You should see the messages:

---------
Crosspeak 1:ASP-_13:HB1 - 1:ASP-_13:HB2 : 0.1064173e+09
---------
Crosspeak 1:ASP-_13:HB1 - 1:ASP-_13:HB2 : 0.226e+00
---------
BKCALC scaler: 470872992

You normally choose a reference peak or a cross peak between protons with fixed distances to get the same scaling for the two spectra. For this lesson, a non-overlapped clean peak belonging to a geminal beta proton pair is a good approximation.

You can also select the reference peak with the cursor. But first you need to zoom in on that peak.

Select the Peaks/Find menu item and set these parameter values:

Find Peak By Name
Action Zoom + Color
Peak name D1 1:ASP-_13:HB1
Peak name D2 1:ASP-_13:HB2

Select OK.

You see the footprint highlighted in yellow in Frame 1. Now you can use the Model/Set Scale menu item. Here you will click the scaling peak.

Select the Model/Set Scale menu item and set Method to via Cursor. Select OK.

Then click the yellow peak in Frame 1.

Now the experimental and theoretical peaks are drawn in green. You can now plot the back-calculated spectrum with the Model/Draw Theoretical menu item, if you want. The back-calculated spectrum is displayed in the middle frame.

During the setup procedure the program connected the experimental and theoretical spectra, therefore changing the limits in one spectrum should trigger the same changes in the other spectrum. To disable or re- enable this behavior, you can use the Preference/Frame Connection menu item.

7.   Compare the two spectra and the structure

Next you will use a command that allows you to click peaks in both displayed spectra, see the two protons highlighted in the structure, and examine the structure.

Select the Model/Show Atoms menu item and move the arrow cursor onto the experimental spectrum. When the large + cursor in each of the spectra appears, click a footprint in the experimental spectrum.

The two protons that correspond to this particular cross peak are labeled in the structure. Also, the display is zoomed - only the atoms around the pair within a 6- radius are shown. At the same time, information about the distance between the two protons is shown in the text window.

Activate the molecule frame (Frame 2) by clicking anywhere on it.

The border color of Frame 2 should change and you should be able to use the real-time 3D display interface. If for some reason you do not have the real-time interface, you can select the Model/Interactive Draw menu item to open the interface. You may rotate, zoom, or translate the molecule to get a better view.

Now first zoom out to find a new peak by pressing the < - > key on the keypad several times, with the cursor in either the experimental or theoretical spectrum frame.

Repeat Step 7 using the Model/Show Atoms command and select a footprint visible in the back-calculated spectrum.

You may use the Model/Blank menu item to get a full view of the molecule.

Type Atoms
Atom Name *
Blank Mode Off

Select OK.

8.   Examine the structure and find cross peaks

First you reset the display to show the entire NOESY spectrum.

Select the View/Limits/Full Limits menu item after activating the experimental spectrum.

This replots both spectra.

Now select the View/Draw Peaks menu item.

Now all footprints are displayed in both spectra.

Select the molecule frame. To get a better view, switch off all atoms by selecting the Model/Blank menu item and setting these parameters:

Blank Mode On
Type Atoms
Atom Name *

Select OK.

Notice that Frame 2: Model is now empty.

Select the Model/Blank menu item again and set these values:

Type Multiple Residues
Residues From 23
Residues To 24
Blank Mode OFF (to see the residues)

Select OK.

Only residues 23 and 24 are displayed in Frame 2.

9.   Select two protons to find the corresponding cross peak

Select the Model/Show Peaks Manual menu item and set these parameter values:

Atom name 1 1:ILE_23:HA
Atom name 2 1:SER_24:HN

Select OK.

The two protons are labeled in the structure, distance information is printed in the text window, and the corresponding cross peaks are highlighted in green:

EXPERIMENTAL --- 218: 1:ILE_23:HA - 1:SER_24:HN : 2.247528
EXPERIMENTAL --- 803: 1:SER_24:HN - 1:ILE_23:HA : 2.247528
------------
THEORETICAL --- 645: 1:ILE_23:HA - 1:SER_24:HN : 2.247528
THEORETICAL --- 703: 1:SER_24:HN - 1:ILE_23:HA : 2.247528
-----------

The second possible way to find a cross peak is to select the protons with the cursor instead of typing their names. This is done with the Model/ Show Peaks Via Cursor menu item.

10.   Exit FELIX

To exit FELIX, select the File/Exit menu item. Toggle off Save Current Session and Save Current Database, then select OK.