Phase Correction in 4D Protein Spectra

The following tutorial refers to the 4D HCNH NOESY spectrum type but the same procedure can be applied for other 4D spectra. Specifically for 4D HCNH NOESY, we need to phase only the F4(PCH0) value. There are several ways to do that. I strongly recommend using the combination of SOLUTIONS 1-3 for certainty.

Table of contents

  1. Solution 1: Phase-Correct the 1st FID

  2. Solution 2: Phase-Correct the Measured 2D Plane of the 4D Spectrum

  3. SOLUTION 3: Phase-Correct the F3-F4 Sum Projection of the 4D Spectrum

  4. SOLUTION 4: Phase-Correct the F3-F4 Projections’ Sum of the 4D Spectrum

  5. Baseline Correction

  6. Credits

Tested environment:

Solution 1: Phase-Correct the 1st FID

Example Data: (13C)HSQC-NOESY-(15N)HSQC on human Carbonic Anhydrase II (850 MHz)

Download example data

  1. Reset all F4 phase values to 0:

    Important: Look at the bottom of the pulse program to find the correct PHC0 and PHC1 values of F1-F3 - if there are any. Read the NUS-reconstruction tutorials for details. Briefly the pulse programs hsqcnoesyhsqccngp4d and noehcnhwg4d_nove require zero values, while sfhmqc_noe_sfhmqc_4Dhcnh.fl the following:

      F4 F3 F2 F1
    PHC0 90 90 -45
    PHC1 0 -180 -180 0

Where P0 is the value you will find after phase correction.

  1. Extract the 1st FID:
  2. Switch to the Frequency Domain:
    • Use ft to switch to the frequency domain.
  3. Phase Correction:
    • Try first the automated 0th order phase correction with apk0 that will try to create an entirely absorptive lineshape in the spectrum.
    • If you are not entirely satisfied with the automatic phasing, enter .ph for manual phase correction. Set the pivot line (right mouse click) near the left limit end to modify only PHC0, not PHC1.

      Note: Adjusting PHC1 (1st order correction) has minimal effect on frequencies near the pivot and no effect on PHC0.
      before phasing

  4. Adjust PHC0:
    • Adjust only PHC0 (0th order correction) so the FID line extends straight and the highest peaks point upwards.

      Note: Avoid using automatic phasing (apk and its variants) as it generally doesn’t work well.
      after phasing

  5. Save and Apply PHC0:
    • Click the Save-disk nD icon to copy the values to the 4D spectrum, then click the Save and Return icon. Issue PHC0 and note down the value (here, -115.0 degrees). Double-click the processed 4D spectrum and ensure all PHC0 and PHC1 values except for F4 (PHC0) are set to 0; unless you used sfhmqc_noe_sfhmqc_4Dhcnh.fl (!).

    correct phase parameters

  6. Initiate NUS-reconstruction:

Solution 2 [MOST ACCURATE]: Phase-Correct the Measured 2D Plane of the 4D Spectrum

Example Data: HN plane of a (13C)HSQC-NOESY-(15N)HSQC on human Carbonic Anhydrase II (850 MHz)

Download example data

This assumes you have measured the F3-F4 (N-HN) plane with the same pulse sequence before recording the whole 4D spectrum.

  1. Prepare the Experiment:
    • Use the F3-F4 plane for phase correction.
    • Run wrpa to create a copy of the whole experiment; name it 61.
  2. Transform the 2D Plane:
    • Open the raw 2D plane in experiment 61 and set all phase values to zero with the following command: 
      4 PHC0 0
4 PHC1 0
3 PHC0 0
3 PHC1 0
2 PHC0 0
2 PHC1 0
1 PHC0 0
1 PHC1 0
    • Run xfb for FT transform.

Whole 34 Plane

whole 34 plane

whole 34 plane

After this, the boundaries of the ROI will appear in STSR and STSI parameters under the PROCPARS tab.

1 ABSF1 1000
1 ABSF2 -1000
1 BC_mod no
2 BC_mod qpol
2 SSB 2.2
2 SI SI*2
1 SI SI*4
  1. Prepare for Manual Phase Correction in the F2 dimension:
    • Enter .ph for manual phase correction.
    • Look for peaks that have an anti-phase component (signals of dispersion character) in the F2 dimension.
    • Place the cursor between the centers of these two peaks. Ensure the cursor is along the line connecting them.
    • Right-click and select “Add”.

Add Peak

Added Peaks

  1. Enter Phase Correction Mode:
    • Click on the icon with the letter “R” to enter phase correction mode for the rows, which correspond to the F2 dimension.
  2. Set the Pivot Point:
    • Right-click at the left limit of the screen and select “Set Pivot”.
    • Place the pivot point away from the peaks to avoid hiding the frequencies we want to phase.
    • Note: Only the PHC0 value will be adjusted, not PHC1, so a pivot line is not necessary.

F2 Before Phasing

  1. Adjust the Phase:
    • Left-button press on the “0” icon and drag your mouse until the strongest signals appear at the top part of each panel.
    • Ensure the frequency lines from end to end look straight and balanced.

F2 After Phasing

  1. Save the PHC0 Value:
    • Click on the “Save-disk” icon to save the selected PHC0 value (e.g., -115 degrees) to the N-HN plane spectrum.
    • Then click the Return icon.
  2. Verify the Phase-Corrected Spectrum:
    • The phase-corrected spectrum should now appear without any anti-phase peak components.

Phased 34 Plane

  1. Save and Apply PHC0:
    • Note down the optimal PHC0 value (e.g., -115.0 degrees) and click the Save-disk nD icon to copy the values to the 2D plane. Then click the Return icon.
    • Transfer the phasing coefficients PHC0 and, if used, PHC1 to the raw 4D (unprocessed) spectrum
  2. Next Steps:

SOLUTION 3: Phase-Correct the F3-F4 sum projection of the 4D Spectrum.

Example Data: Raw_Spectra/Carbonic_Anhydrase_2/hCA2-wt_850/9

Download example data

  1. Spectrum processing without NUS reconstruction:
    • Reset all phase values to 0 as described in SOLUTION 1, step 1, unless you:
    • used sfhmqc_noe_sfhmqc_4Dhcnh.fl pulse program (see NUS-reconstruction tutorials)
    • you have already found a F4(PHC0) value using SOLUTION 1, and now you want to optimize it further with this SOLUTION. - Typically, you should perform NUS reconstruction of the 4D spectrum using the ftnd 0 command. However, this process can take anywhere from 3 to 12 hours, which may not always be practical. In some cases, you might choose to skip the full NUS reconstruction. By running the command ftnd 0 nusft, you can process the full spectrum-including fid baseline correction, window multiplication, Fourier transform, and phase correction—without reconstructing the missing data points. Use this option at your own discretion. While this method is faster than a full NUS reconstruction, it still requires time and may result in a lower-quality spectrum. This is especially true for 4D spectra, which typically use low NusAMOUNT values (less than 3%). If you find the quality insufficient for the next steps, you can reprocess the 4D spectrum using the full ftnd 0 command.
  2. Extract Sum Projection:
    • Use the command sumpl 34 all all 34340 to extract the sum 34 projection, which is the sum of positive and negative 34 projections. It looks quite noisy with dominant negative signals, and barely resembles the 34 plane that we phased in SOLUTION 2. But that’s OK.

Sum 34 Projection

  1. Create Imaginary Dimensions:
    • Run xht1 and xht2 on the 34 sum projection to create the imaginary dimensions.
  2. Manual Phase Correction:
    • Our aim is to suppress the negative signals as much as possible. For some types of spectra this doesn’t imply that they are correctly phased, but for 4D HCNH NOESY it does.
    • Run .ph for manual phase correction and phase correct only on F4, as described in SOLUTION 2, steps 3-10. The only difference here is that we are processing 2D spectrum derived from a 4D spectrum measured with NUS, and hence we cannot do zero-filling or truncate the dimensions with xfb command. We can only do abs2 and abs1 for baseline correction.
    • We can also manually apply different phase values (PHC0, PHC1) in F2 using the command xf2p.
    • Identify and select peaks with antiphase component that span as much of the spectrum as possible. Not many available in this one.

sumpl 34 Selected Peaks

sumpl 34 Before Phasing

sumpl 34 After Phasing

sumpl 34 Phased

  1. Apply Phase Correction to 4D:
    • Copy the F4(PHC0) value to the 4D spectrum.
    • Execute pknd 4 (WARNING: very slow!; may be faster to redo ftnd 0 or ftnd 4)

SOLUTION 4: Phase-Correct the F3-F4 projections’ sum of the 4D Spectrum.

Warning: This solution is not the most accurate one. Is otherwise identical to SOLUTION 3, but instead of creating the sum projection, we create the positive and negative projections, and then we sum them.

Example Data: Raw_Spectra/Carbonic_Anhydrase_2/hCA2-wt_850/9

Download example data

  1. Spectrum processing without NUS reconstruction:
    • Reset all F4 phase values to 0 as described in SOLUTION 1, step 1.
    • Initiate raw spectrum processing with zero phase values as described in SOLUTION 1, step 2.
  2. Extract Projections:
    • Use the command projplp 34 all all 34 to extract the positive 34 projection.

Positive 34 Projection

Negative 34 Projection

  1. Create Imaginary Dimensions:
    • Run xht1 and xht2 on the 34 projections to create the imaginary dimensions.
  2. Sum Positive and Negative Projection
    • Open 34 and type wrp to copy it to a new file named 343409.
    • Run adsu, and from the new menu bar, go to Add -> Change constant ALPHA and set it to 1.
    • Then, go to Add -> Change constant GAMMA and set it to 1.
    • Select Define 2nd Dataset and type the name of the negative projection (340).
    • Execute the add2d command to add the negative projection (340) to the copy of the positive (34340).

Sum of Positive and Negative 34 Projections

  1. Manual Phase Correction:
    • Our aim is to suppress the negative signals as much as possible. For some types of spectra this doesn’t imply that they are correctly phased, but for 4D HCNH NOESY it does.
    • Run .ph for manual phase correction and phase correct only on F4, as described in SOLUTION 2, steps 3-10. The only difference here is that we are processing 2D spectrum derived from a 4D spectrum measured with NUS, and hence we cannot do zero-filling or truncate the dimensions with xfb command. We can only do abs2 and abs1 for baseline correction.
    • We can also manually apply different phase values (PHC0, PHC1) in F2 using the command xf2p.
    • Identify and select peaks that span the entire spectrum.

add2d Selected Peaks

add2d Before Phasing

add2d After Phasing

add2d Phased

  1. Apply Phase Correction to 4D:
    • Copy the F4(PHC0) value to the 4D spectrum.
    • Execute pknd 4 (WARNING: very slow!; may be faster to redo ftnd 0 or ftnd 4)

Baseline Correction

Baseline correction does not have a significant effect on 4D spectra. It should be considered a final touch that does not affect the peak shape.

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