3D data processing


About the lesson

In this lesson you learn the basic processing steps involved in transforming 3D time-domain data to frequency-domain data. The lesson leads you through the processing and basic display a 2D slice for a 3D data set.

This lesson takes approximately 30 minutes to complete once you are comfortable with the procedures.


Lesson 1: 3D Data processing and display

1.   Setting up for the lesson

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 3D\Lesson1 folder.

The sample data set is an HNCO experiment on ubiquitin, a 76-residue protein; the unprocessed data (the fid and procpar files) should be located in the Kelix_Practice\tutorial directory before you start.

2.   Starting FELIX

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\3D\Lesson1\ using the Preference/Directory... command. Create a new database by opening the File/New... command and choosing Create a new matrix or DBA file. Make sure the File Type is set to DBA(*.dba) and give the database a new file name, such as test3d. Click OK.

3.   Transforming the data

Select the ProcessND/Open and Process 3D menu item.

In the SELECT SPECTRUM FILE control panel, make sure the Filter Type is set to All Files, then select fid from the Files list and click OK.

The second control panel displays the header parameters. This is Varian States 3D data, which should have the following default parameters:

D1 Parameters
Data Size 512
Spectrometer Frequency 599.945
Sweep Width 8000.001
D2 Parameters
Data Size 32
Spectrometer Frequency 150.869
Sweep Width 2431.88
D3 Parameters
Data Size 50
Spectrometer Frequency 60.797
Sweep Width 3000.075
Environment
Pulse Program gCT_hn_co
Solvent D2O
Temperature 303.0
Data Source Varian

Once you have verified that all the parameters are set correctly, click OK.

The third control panel now displays the acquisition parameters.

The Acquisition Parameters should be set like this:

Data Type Complex
Acquisition Mode States or States-TPPI
First Incremented d3, d2
Quartet Order phase2, phase

Once you have verified that all the parameters are set correctly, click OK.

In the fourth control panel, enter these parameter values:

Dimension To Process D1 FT
Output Matrix Filename hnco.mat
Dimension 1 Size 512
Dimension 2 Size 64
Dimension 3 Size 128
Correct DC-offset on
Fraction 0.5
Correct 1-st point None
Solvent Suppression None
Window Function Sinebell
FT Type Complex
Phasing Mode Interactive
FID to phase 1
Baseline correction None
Reverse Vector off
Extract Half Spectrum Left Half
Output Level Verbose

Click OK.

Note: The processed data will be saved in a FELIX matrix file named hnco.mat in this example. This file will be located in the folder designated for FELIX matrices. To view or change that folder, select Preference/Directory before 3D processing.
If you want to specify the folder interactively, you can click the button next to Output Matrix File and navigate to the desired folder and specify a filename.

In the Sinebell Parameters control panel, verify that these parameters are set:

In the Sinebell Parameters control panel, verify that these parameters are set:

Method: Parameter
Data Size: 512
Phase Shift: 90.0

Click OK.

FELIX displays the real-time phasing interface.

Adjust phase0 (and phase1, if necessary). Click OK when you are done phasing.

Next FELIX builds the matrix.

When FELIX prompts you to initiate the transform, click OK.

When the processing of the D1 dimension has finished, FELIX should displays the message:

     D1(t3) transform completed.

Elapsed time was 40 seconds

In this tutorial we continue the processing with the second dimension.

4.   Transforming the D2 dimension

In the control panel, enter these parameter values:

Dimension To Process: D2 FT
Load Matrix in Memory: off
Processing Mode: Bundle
Correct 1st-point: None
Window function: Sinebell
Linear Prediction: off
FT Type: Complex
Phasing Mode: Use Parameters
Phase0: 0
Phase1: 0
Baseline Correction: None
Reverse Vector: off
Output Level: Verbose

Click OK.

In the next control panel, enter Parameter, 32, and 90 for the Sinebell Window parameters. Click OK to start the D2 transform.

In the output window, FELIX reports that there are 65536 D2 vectors to process. This is 512 x 128, or D1 x D3.

When FELIX has finished processing the D2 dimension you can continue with the third dimension.

In the next control panel enter these parameter values:

Dimension To Process: D3 FT
Load Matrix in Memory: off
Processing Mode: Bundle
Correct 1st-point: None
Window function: Sinebell
Linear Prediction: off
FT Type: Complex
Phasing Mode: Use Parameters
Phase0: 0.0
Phase1: 0.0
Baseline Correction: None
Reverse Vector: off
Output Level: Verbose

Click OK.

In the control panel, enter Parameter, 50, and 90 for the Sinebell Window parameters. Click OK to start the D3 transform.

Now FELIX reports that there are 32768 D2 vectors to process. This is 512 x 64, or D1 x D2.

When FELIX has finished processing the D3 dimension, again open the matrix and display it.

5.   Reading in the matrix

Select the File/Open menu item or click the Open icon.

File Type: Matrix
Filename: hnco.mat
Access: Read only
Storage: Keep on Disk

Click OK.

FELIX calculates the contour level automatically before displaying the spectrum. You can click the Increase Threshold or Decrease Threshold icon to adjust it.

Move the slider in the REAL-TIME PLANE dialog box to view other D1-D2 planes. You can also use the left or right arrow key to step through the planes when the slider is highlighted. Click OK to close it.

Click the Orthogonal 1D Slice icon, then click the cursor on a peak to view the D3 slice. Select the Contour option from the combo box in the toolbar to display the 3D spectrum again. You can repeat this step several times to check the D3 slices for more peaks. You should notice that D3 dimension is off phase.

6.   Automatic phase correction in D3 dimension

Make sure the 3D spectrum is displayed. Select the Contour option from the combo box in the toolbar if necessary.

Select the ProcessND/Phase Correct menu item. In the Parameters control panel, enter these parameter values:

Rephase: D3 vectors
Phasing Mode: Automatic

Click OK.

In the next control panel, check #1. Click the Cursor button. Drag the cursor so that the rubber-band box covers the upper field solvent peaks. After that, the control panel should display a range close to From 451 to 512. Click OK.

This excludes the solvent peaks from being considered while automatic phasing using the PAMPAS algorithm (Dzakula, 2000). It takes about half a minute to search all the D3 vectors for test peaks, calculate the phase errors, and then apply the phase correction to the whole matrix.

7.   Picking the 3D peaks

Select the Peaks/Pick All menu item. In the Parameters control panel, enter these parameter values:

Select the Peaks/Pick All menu item. In the Parameters control panel, enter these parameter values:

Peak Halfwidth
D1 Minimum: 1.1
D2 Minimum: 0.7
D3 Minimum: 1.1

Leave all other parameters at their current values and click OK.

When the peak picking finishes, a spreadsheet appears with the picked peaks.

8.   Coloring peaks by slices

Select the Preference/Peak Display menu item. In the PEAK DISPLAY PREFERENCE control panel, select Item # as Label Peaks, change Label Size to 0.15 (inch), and set Coloring Mode to By Slice. Click the Set button.

In the COLOR PEAKS BY PLANE control panel, leave the default values and click OK. Click the Plot icon from the tool bar to redraw the spectrum.

Click the Real-Time Plane Selection icon. Move the slider in the control panel to step through the planes.

The peaks are displayed in different colors, depending on whether they are centered on the current plane or not.

9.   Plotting 2D slices of the 3D spectrum

Select the View/Limits/Select Plane menu item. In the control panel, set the Unit to points and set D1_H1-D3_N15 at D2_C13 to 32.

Click OK.

Now you have D1 as the horizontal axis and D3 as the vertical axis. The number of the D2 slice, 32 (or the chemical shift value if you have selected PPM as the axis unit) is shown in the lower-right corner.

You can use the Preference/Plot Parameters menu item to adjust the attributes of the plot, if necessary, and to select the appropriate display parameters, such as Contour Threshold.

Next you learn to page through the 2D slices interactively.

If the Real-time Plane control is not displayed yet, click the Real-time Plane Selection icon to display it. Click the slider in the Real-time Plane control and move it, then release it. You can also step through the planes by pressing the left or right arrow key when the slider is highlighted.

This updates the contour plot. You can also type in a new plane via plane number or by using the ppm box

10.   Exiting FELIX

At this point you may exit FELIX by selecting the File/Exit menu item.