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:
This lesson demonstrates two uses of the Model module.
1. Setting up for the lesson
2. Starting FELIX and opening the model database
This database file contains all the information you need in this tutorial.
3. Open the experimental matrix
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
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.
Multiple cross peaks appear.
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
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.
You see a message in the text window:
and after a short while:
Next you choose a scaling peak. This peak should have the same intensity in both the experimental and back-calculated spectra.
You select this peak to have the same intensities in the two spectra. You should see the messages:
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.
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.
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.
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-Å radius are shown. At the same time, information about the distance between the two protons is shown in the text window.
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.
Repeat Step 7 using the Model/Show Atoms command and select a footprint visible in the back-calculated spectrum.
8. Examine the structure and find cross peaks
First you reset the display to show the entire NOESY spectrum.
This replots both spectra.
Now all footprints are displayed in both spectra.
Notice that Frame 2: Model is now empty.
Only residues 23 and 24 are displayed in Frame 2.
9. Select two protons to find the corresponding cross peak
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:
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