3D data processing

Lesson 1: 3D data processing and display

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 (of a 3D cube and a 2D slice) for a 3D data set.

Topics in this lesson

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

1.   Setting up for the lesson

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 your current working directory before you start.

At the UNIX prompt, issue the command

Copy the fid and procpar files to the directory where you will be running FELIX:

2.   Starting FELIX

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

3.   Transforming the data

Since you will be working with a 3D Varian file, simply 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 select 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, select 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, select 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

Select OK.

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

Method: Parameter
Data Size: 512
Phase Shift: 90.0

Select OK.

FELIX now automatically puts you into the real-time phasing interface.

Adjust phase0 (and phase1, if necessary). Click Keep when you are done phasing, then select OK to begin processing the D1 dimension.

Next FELIX builds the matrix.

When FELIX asks if you want to initiate the transform, answer OK.

When the processing of the D1 dimension has finished, you should get the message: 

     D1(t3) transform completed.

     Elapsed time was 40 seconds

It is a good idea to look at the matrix so far; if you have not begun the processing correctly you can often detect errors at this stage.

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

Select OK.

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

You are told 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

Select OK.

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

Now you are told 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.

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

Select OK.

FELIX calculates the contour level. Now you need to raise this contour level.

Click the Increase Threshold icon.

Use the Real-time Plane selection control to view the D1-D2 planes.

Now you can view a 3D cube display.

First select the Preference/Plot Parameters menu item and choose only one contour level with these parameters:

Contour Threshold: 0.1
Scheme: Green
Number of Levels: 1
Negative Levels: off

Select Set.

When you select a Ramp Type color scheme, the Number Of Levels parameter is fixed and cannot be changed. If you select an individual color (such as Green) or a user-defined color scheme, you can once again change the Number Of Levels parameter.

Next select the View/Limits/Manual Limits menu item and enter the following parameter values:

Limit Type: Points

D1_H lower: 1
D1_H upper: 500

D2_C13 lower: 1
D2_C13 upper: 64

D3_N15 lower: 1
D3_N15 upper: 128

Select OK to draw the spectrum.

Select the View/Plottype/3D Contour Plot menu item to view the contour plot.

Select the View/Plottype/Interactive menu item to activate the 3D viewing interface and rotate the spectrum. Select Exit when you are finished.

6.   Picking the 3D peaks

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 select OK.

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

Select the View/Plottype/3D Contour, then the View/Plottype/Interactive menu items to bring up the rotation menus again. Rotate the object and verify that the peaks have been picked in 3D.

Select Exit to leave the Interactive Draw control panel.

7.   Plotting 2D slices of the 3D spectrum

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

Select OK.

Now you have D1 as the horizontal axis and D3 as the vertical axis. The number of the D2 slice, 32, 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.

Click the slider in the Real-time Plane control and move it, then release it.

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

At this point you may exit FELIX by selecting the File/Exit menu item or you can continue on to the next lesson, being sure to first use the File/Close Matrix menu item to close the matrix.