In Lesson 1: 2D processing, display, and analysis, you will learn to process, display, and analyze 2D matrix files.
In Lesson 2: Analyzing Relaxation Data, you will learn to perform a relaxation analysis.
In this lesson you process a 2D matrix, display the spectrum using a variety of standard plotting methods, and assign the cross peaks. You start by processing a 2D matrix using the EZ macros.
This lesson takes approximately 60 minutes to complete once you are comfortable with the procedures.
If not done yet, set up the tutorial files as described in "Setting up tutorial files" on page viii.
The files for this lesson are located in the 2D\Lesson1 folder.
When FELIX processes data it expects to see the data with the same directory structure as existed on the spectrometer. So the whole 2D\Lesson1 folder must be copied to keep the directory structure.
Start FELIX by double clicking the Felix icon on your desktop, or by clicking the Start button on the Windows taskbar, then selecting Programs/Accelrys Felix 2004/Felix 2004.
If FELIX prompts you to restore from last session, click Cancel.
Change your Current Working Directory to C:\Felix_Practice\2D\Lesson1\ using the Preference/Directory... command. To start a new database, select File/New... and choose Create a new matrix or DBA file. Make sure the File Type is set to DBA(*.dba) and give the database a new filename, such as test. Click OK.
Where you create or open the .dba file will determine your working directory. To change the working folder, select Preference/Directory.
FELIX displays the first FID of a 2D HSQC spectrum collected on a Bruker spectrometer.
Instead of going directly to 2D processing, first we will apply a few 1D processing functions on the displayed FID in order to get some spectral parameters.
FELIX displays the FID along with the apodization function, colored red. You may experiment with different settings of the wsize and wshift parameters, and watch their effect on the FID and the processed spectrum.
FELIX displays the apodized FID on the screen.
6. Phase-correcting the spectrum
Select the Process1D/Phase Correction command. In the control panel, select the Real-Time option for Method and click OK.
Click the Pivot button in the REAL-TIME PHASING dialog, and click a peak in spectrum which you would like to use for zero-order phasing. Using the first slider to adjust the Phase0 parameter to phase this peak, then adjust the Phase1 parameter as necessary.
Since this is a modeless dialog, you can activate the main commands or the toolbar icons to change the spectral display when you are phasing the spectrum.
Now that you have a rough idea of the apodization and phase correction parameters, you can proceed with processing the D1(t2) dimension of the 2D data set.
Record the apodization and phase correction parameters on paper. The FELIX symbols that store these parameter may get overwritten in the following steps.
Caution
You will now open the ser file again to process a 2D spectrum.
7. Processing the D1 (t2) dimension of the 2D data set
Select the ProcessND/Open and Process 2D... command. In the first control panel, select the previous ser file. In the next control panel, leave most of the header parameters at their default values (read from the spectrometer header files), except for these:
In the next control panel set the following parameter values:
In the next control panel, set the following parameter values:
The processed data will be saved in a FELIX matrix file named bruker.mat in this example. This file will be located in the folder designated for FELIX matrices. To change that folder, interactively, press the ... button next to Output Matrix Filename and navigate to the folder you want.
As the D1 transform proceeds, the progress is illustrated in the status bar. This step is often completed in less than one second.
After the first dimension is processed, FELIX shows the control panel for processing the second dimension.
8. Processing the D2 (t1) dimension
Set the following parameter values in the control panel:
FELIX usually completes this step in a few seconds.
When processing completes,FELIX opens the matrix since you chose to display the matrix at completion. The contour threshold is calculated, and the matrix is then displayed.
FELIX calculates the plot levels. You can change them manually
.Select the Preference/Plot Parameters command (or click the Plot Parameters icon in the toolbar). Set the following values:
Select the Intensity or Contour options from the combo box in the toolbar to display the intensity map or contours of the spectrum, respectively.
Click the Autoscale icon in the toolbar to toggle between the proportional mode and fill-in mode.
Click the Increase Threshold and Decrease Threshold icons in the toolbar to raise or lower the threshold for spectral display.
While displaying the spectrum, the data are always read from the matrix, not from a graphics file, so that display parameters can be recalculated at any time. Hence, FELIX redraws the graph with each plot command.
At this point you could examine the D1 and D2 vectors in more detail to determine whether further baseline correction or phasing adjustments are necessary.
Referencing of the matrix happens automatically, since the header parameters are read or adjusted during processing. You can further adjust the referencing, for example, by giving more descriptive names for the axis.
Select the Preference/Reference command. In the control panel, leave every parameter at its current value, except for these:
The 2D spectrum should now be redisplayed with the correct referencing for each axis.
11. Viewing an expanded-region contour plot
You can choose expanded regions with the cursor or by inputting numeric parameters:
When you release the mouse button, the region selected expands to fill the window. The plot is still in intensity mode, hence the contour levels are not shown.
In the control panel that appears, the parameters are filled in with the values of the current plot.
FELIX displays the new 2D region.
You can save these parameters and reuse them for other plots; for example, if you were analyzing a series of spectra collected with different mixing times and always wanted to observe identical regions.
FELIX now plots the 2D matrix in contour mode, with a color-coded intensity scale.
12. Changing the 2D drawing parameters
If no peaks are visible, try decreasing the contour level to cut lower into the spectrum. If the peaks are outlined but you do not see the circles shrinking to define the tops of the resonances, try increasing the level multiplier to increase the space between levels:
FELIX displays three grid lines between each pair of major tick marks.
14. Returning to the full spectrum
FELIX displays red boxes around all cross peaks meeting the criteria defined in the control panel.
A new window containing the peak table is displayed to the left of the spectral window. Note that, by default, whenever a new window (table or spectral) is open, FELIX automatically re-arranges the layout of the windows. You can turn off this feature by selecting Preference/Frame Layout from the main menu and set Action to None. You can also do the automatic re-arrangement at anytime by selecting Window/Auto Arrange.
Note: When one or more table windows are open, only the menu and tool bar of the currently activated window are visible. If you want to select a certain menu item or tool bar icon, be sure to click the corresponding window first to activate its menu and tool bar (if any).
16. Deleting peaks and replacing them
Activate the spectral window by clicking on it. Select the Peaks/Remove One command (or right click inside the spectral window and select Remove One Peak in the context menu). The cursor becomes a +. Click one or two red boxes to remove them, then click in the empty space to the left of the frames to return to a normal cursor.
Click the Zoom icon in the toolbar and zoom into a few peaks. Select the Peaks/Edit command (or right lick side the spectral window and select Edit One Peak in the context menu) to manually adjust the box defining a peak.
Click in the center of a red box of a picked peak. The box becomes green, indicating that it is selected for editing. Click the center of the box and drag to move the entire box or click near a corner of the box and drag to resize it.
To leave editing mode, press the <Esc> key while the spectral window is activated.
Select the Peaks/Name One Peak command and click one of the red boxes.
Set the following parameters to assign the resonance:
Click OK, then press the <Esc> key to return to a normal cursor.
You do not need to assign the peak in both dimensions. You can label the cross peaks in one dimension at a time, as the assignment is made. This is usually how assignments are observed.
If you want to use the restraints list directly in the Insight II or Discover program, you have to use the Insight II proton names as the peak names. At the moment there is no check of atom names, so you may enter anything you want.
FELIX searches the assignment list, and colors the boxes surrounding all peaks with a label of h1 in D1 dimension in yellow. FELIX reports the coordinates of the peak on the status bar.
To calculate the volumes of the picked peaks, select the Measure/Integral/Volume command. In the control panel, select the Measure All Volume option for Action. In the next control panel, leave the Peak and Volume set at their defaults. Set Volume Slot Number to 1 and Mixing Time to 0.1. Click OK.
FELIX displays the BUILDING NEW VOLUME ENTITY control panel; set Total Mixing Time Slots to 6 and click OK.
You can now view the volume data with the Edit/Table command (selecting the vol:volumes entity) or write the volume data to a file with the File/Export/Table command from the main menu.
To calculate restraints from these volumes based on the two-spin approximation, you must open or create a scalar entity for the database, define a scalar pair, create the restraints (strong-medium-weak, or any other listed choice), and write the restraints file. The appropriate commands are in the Measure menu.
In this lesson you learn to perform a relaxation analysis based on analyzing heteronuclear relaxation data. It is assumed that R1, R2, and heteronuclear NOE were measured as a series of 2D HSQC (or equivalent) spectra. The data used are parts of the relevant spectra acquired for apocalbindin D9k (Akke et al. 1993).
See "Setting up tutorial files" on page viii as needed.
Start FELIX by double clicking the Felix icon on your desktop, or by clicking the Start button on the Windows taskbar, then selecting Programs/Accelrys Felix 2004/Felix 2004.
If FELIX prompts you to restore from last session, click Cancel.
Change your Current Working Directory to C:\Felix_Practice\2D\Lesson2\ using the Preference/Directory... command. Open the relax.dba database using File/Open... Make sure the File Type is set to DBA (*.dba). Click OK.
The name and path of the database file appears on the title bar of FELIX main window.
3. Measure peak heights in the R1 series of spectra
Select the Measure/Relaxation/Measure Heights/Volumes... command. In the control panel, set these values:
Select the Measure/Relaxation/Measure Heights/Volumes... command. In the control panel, set these values:
In the next control panel, FELIX prompts for the first spectrum in the series and some parameters.
Select the t1_1.mat matrix and set these values:
FELIX plots the spectrum, repositions the peaks to their exact centers. and calculates the peak heights.
When the spectrum selection control panel appears again, you need to specify file names and parameters for the remaining spectra in the same way as you did above.
FELIX displays the relaxation delays in the last row of the table.
4. Evaluate the signal/noise ratio for the peak heights
Select the Signal/Noise Ratio... command in the Relaxation menu. In the control panel, enter:
FELIX calculates the peak heights in this duplicate spectrum, calculates the average height difference between this spectrum and its twin spectrum, and derives the uncertainty of the volume determination.
FELIX reports these values in the text-prompt window. If you have more than one duplicate time point in your relaxation series, the uncertainties for the other time points are interpolated or extrapolated. For a single duplicate measurement, the uncertainties are promoted to the other points.
FELIX displays the uncertainties in the last row of the table.
FELIX plots a graph of the peak height vs. relaxation delay, including error bars. Due to the good S/N in the spectra, the error bars may not be immediately apparent.
FELIX displays the corresponding time course or informs you that no such peak number exists and lets you try again.
FELIX uses data from the currently active tables to display the time courses. If you want to view data from different tables, use the Preference/Table... command to assign another relaxation table.
6. Fit R1 values to the time courses
FELIX now fits the time course data to the exponential function:
| Eq. 1 |
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and derives the relaxation rate R1 from the coefficient a2 in the exponent. FELIX reports the relaxation rate R1, its standard deviation, and the c2 value for each fit in the text window.
FELIX stores the following in the table rel:r1: relaxation rate R1, the raw coefficients for the offset a0 and linear term a1 in the function, and their standard deviations.
In addition, FELIX stores the c2 value of the fit for each time course.
If you now view a time course, FELIX plots the fitted function in red, along with the experimental peak heights. This lets you visually judge the quality of the fit. You can print the plot by clicking the Print icon.
The t2_1b.mat is a duplicate spectrum. R2 time courses are fitted to the simple exponential function:
| Eq. 2 |
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and the general exponential function:
| Eq. 3 |
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Whichever function yields the lower c2 value is used to derive the R2 relaxation rate. FELIX reports the R2 value, its standard deviation, and the c2 value in the text window and also tells you which function was used.
All the fitted values are stored in the table rel:r2, analogous to the R1 data (see Step 6).
8. Evaluate heteronuclear NOEs
FELIX plots the first spectrum, repositions peaks to their exact centers, and measures peak heights. Then it plots the second spectrum and measures peak height.
In the fifth control panel enter:
FELIX plots the first duplicate spectrum, repositions peaks to their exact centers, and measures peak heights. Then it plots the second spectrum and measures peak heights. Finally it reports the NOEs and their standard deviations to the text window and stores them in the table rel:noe.
Once you have all R1, R2, and NOE values evaluated and stored in the database, you can generate an input file for the Modelfree program (A. G. Palmer, Columbia University, http://www.hhmi.columbia.edu/ palmer/).
Select Modelfree Input from the Relaxation menu. Enter the following values in the control panel:
Now you have initial input files for the tmest and Modelfree programs. For more information about working with Modelfree, please refer to its documentation and to the scientific literature.
