Peak Picking in 4D Spectrum with POKY

Overview

The general workflow of this tutorial starts with referencing and converting the spectra to POKY/Sparky format. Then, we will create 2D projections from the 4D spectrum, which will help us reference it properly.

For peak picking, we will follow a systematic strategy that, although more involved, will allow for higher precision in identifying peaks in the 4D spectrum while minimizing noise. Since the 2D projections are derived directly from the 4D spectrum, they provide a more accurate reference than the HSQC spectra, whose peak centers may deviate slightly from those in the projections.

To ensure accurate peak selection, we will use the 2D projections as intermediate reference points for restricted peak picking in the 4D spectrum. The workflow is as follows:

1. Overlay the projections onto the corresponding HSQC spectra
2. Identify peak centers by using the HSQC spectra as references
3. Use these peak centers to perform restricted peak picking in the 4D spectrum
4. Unfold or unalias peaks as necessary
5. Remove noise peaks from the 4D spectrum

By following this approach, we ensure that the final set of picked peaks in the 4D spectrum is as accurate and noise-free as possible.

Prerequisites

• Installation of POKY or NMRFAM-Sparky; license for POKY.
• Copy the enhanced Restricted Peak Pick POKY plugin that reads the tolerance values from the peak’s “Note” field from nmr_tutorials/SPARKY_and_POKY/POKY/scripts/restrictedpick.py to your POKY/poky_linux/modules/poky/ folder.
• Access to the specified CA2 protein 4D HCNH NOESY, 15N HSQC and 13C HSQC spectrum files.

Steps

---

Step 1. Reference the HSQC Spectra in Topspin

Follow the instructions to reference the 1H-15N and 1H-13C HSQC in Topspin with BioTop.

---

2.2 Convert Spectra to UCSF Format

Enter the directory where each spectrum is saved in Bruker format and run bruk2ucsf from there—running it from another directory will fail.
For example, to convert the 1H-15N, 1H-13C HSQC spectra, and the 4D HCNH NOESY:

 bruk2ucsf_run 6/pdata/1/2rr /srv/NMR/Peak_Picking/Nanoluc/15N_HSQC.ucsf
 bruk2ucsf_run 7/pdata/1/2rr /srv/NMR/Peak_Picking/Nanoluc/13C_HSQC.ucsf
 bruk2ucsf_run 5/pdata/1/4rrr /srv/NMR/Peak_Picking/Nanoluc/4D_HCNH_NOESY.ucsf

Note: You can also convert the spectra from Bruker to UCSF format in POKY/Sparky, but you cannot rename the axes in that process.

2.3 Rename Axes

Rename the axes in the 1H-15N and 1H-13C HSQC spectra:

ucsfdata -a1 N -a2 HN 15N_HSQC.ucsf
ucsfdata -a1 C -a2 HC 13C_HSQC.ucsf

Print the axis values of the 4D HCNH NOESY:

ucsfdata 4D_HCNH_NOESY.ucsf

Example output:

axis                          w1          w2          w3          w4
nucleus                       1H         13C         15N          1H
matrix size                  256         256         256         416
block size                     8           8           8          13
upfield ppm                1.194       6.301     101.402       5.279
downfield ppm              8.208      73.001     133.002      10.622
spectrum width Hz       6666.667   15939.978    3043.445    5078.125
transmitter MHz          950.374     238.980      96.311     950.374

From the upfield and downfield rows, you can guess which axis is HC and which is HN. In this example, the following command renames them properly—amidic protons have higher shift values than the aliphatic protons:

ucsfdata -a1 HC -a2 C -a3 N -a4 HN 4D_HCNH_NOESY.ucsf

IMPORTANT: Make sure that axes are named consistently in all spectra; otherwise, you will encounter problems during peak picking.

2.4 Create C-HC and N-HN Projections

For a detailed tutorial, see Create_2D_projections_from_4D_spectrum. Briefly, extract the N-HN projection from the 4D HCNH NOESY. You may need to adjust the -p[1-4] values according to your 4D spectrum dimension order:

ucsfdata -p1 -r -o C-N-HN.ucsf 4D_HCNH_NOESY.ucsf
ucsfdata -p1 -r -o 2D_N-HN_proj.ucsf C-N-HN.ucsf

Similarly, for the C-HC projection:

ucsfdata -p4 -r -o HC-C-N.ucsf 4D_HCNH_NOESY.ucsf
ucsfdata -p3 -r -o 2D_HC-C_proj.ucsf HC-C-N.ucsf

---

Step 3. Loading the Spectra

Load the spectra

• Open the UCSF files with the fo command (make sure to display Poky Spectrum type of files in the pop-up browser), navigate to the folder, and select your spectra. Alternatively, you can copy the full path to each spectrum (for example, realpath 4D_HCNH_NOESY.ucsf in the Shell) and paste it into the pop-up browser.
• Do the same with the 2D projections and the HSQC spectra.
• Use xa to show the nucleus types on the axes., xr to roll the axes and xx to transpose them.
• Fix the aspect ratio by hitting vt and increasing the Aspect (ppm) for example to 12, and then Apply.

---

Step 4. Adjusting the Spectra

Synchronize Spectra

• Click yt to synchronize the N axes of the 1H-15N HSQC and 4D_HCNH_NOESY first, and then synchronize the HN axes of those same spectra. Remember to synchronize one axis at a time!
• Do the same for the 1H-13C HSQC and 4D_HCNH_NOESY, and then for both HSQC spectra and the respective 2D projections.

Correct the contour levels and colors

• Type vC to bring up the contour level control scrollbars and adjust the contour levels.
• Type ec to bring up the easy contour dialog allowing you to adjust all loaded spectra, including their colors.
• If you double-click on a spectrum in the dialog it will open up the contour level dialog (equivalent to the ct command).

Align the 2D_N-HN_proj to the 15N_HSQC

• The 4D axes are usually completely off and must be aligned to the reference HSQC spectra. To achieve this, use the 2D projections.
• Hit ol to overlay 2D_N-HN_proj.ucsf onto 15N_HSQC.
• You may reduce the contour number to 1 for one of the two spectra for better visibility. Also hit oz to increse the contour thickness and the peak marker thickness.
• Manually pick the most “trustworthy” peak in the 15N_HSQC (F8 to enter peak picking mode, F1 to exit it) and find the same peak in the 4D.
• Type al, and in the pop-up:
  • Set Align spectrum to 2D_N-HN_proj
  • Set using peak in to 15N_HSQC
  • The axes should match, thanks to the renaming we did earlier.
  • Now hold the Shift key and select one “trustworthy” peak in each spectrum for alignment.
  • Hit Align.
  • Alternatively pick a set of matching peaks (button F8) in both spectra and click the Auto align option (slower and doesn’t always work well).

Align the 2D_HC-C_proj to the 13C_HSQC
Follow the same procedure described in the previous step.

Reference the 4D_HCNH_NOESY

• Hit st and copy the shift values from the aligned 2D_N-HN_proj to 4D_HCNH_NOESY.
• Make sure that every time you copy a shift value from one spectrum to the other, you click Apply, or else the value is not saved.
• Similarly, copy the shift values from the aligned 2D_HC-C_proj to 4D_HCNH_NOESY.
• When you finish, click OK, and the 4D_HCNH_NOESY will be referenced!

---

Step 5. Peak Picking

Adjusting Contour Levels and Preparing Reference Peaks

• Adjust the contour levels (both positive and negative) in the 15N HSQC spectrum to optimize peak visibility.
• Press F8 to enter peak picking mode and select all visible peaks.
• Use the following Python function to estimate the expected number of N-H (in-phase) peaks from the amino acid sequence in the multiplicity edited 15N HSQC:

  def estimate_15N_hsqc_peaks(sequence: str) -> int:
    # Count backbone amides = total residues minus any prolines and the N-terminus
    backbone_peaks = sum(1 for aa in sequence if aa != 'P') -1
    # Count side-chain peaks from R (NE–HE), K (NZ–QZ), W (NE1–HE1), and H (ND1-HD1)
    sidechain_peaks = sequence.count('R') + sequence.count('K') + sequence.count('W') + sequence.count('H')
    return backbone_peaks + sidechain_peaks
  

• Gradually increase or decrease the contour levels until the number of in-phase (positive intensity) picked peaks is approximately the expected number of N-H peaks (292 for CA2 protein). I set the contour level to 1.4e+06, which captured 274 in-phase peaks, as below that a lot of noise peaks were emerging.
• Press lt to open the peak list and export the peak list from the 15N HSQC spectrum by saving it to a file.
• Switch to the 2D N-HN projection, hit ol and overlay 15N_HSQC to 2D_N-HN_proj.
• Delete all current peaks from the 2D N-HN projection.
• Press rp to load the saved peak list from the 15N HSQC. Alternatively, just copy (Ctrl+c)-paste (Ctrl+v) from one spectrum to the other.
• Press pa to select all peaks.
• Press pc to adjust the peak positions to the contour centers of the 2D projection.
• Go through all the peak markers, visualize them and adjust their position manually to be at the center of the contours hills in the 2D_N-HN_proj. To help you spot the overlapping peak markers, hit lt, sort by frequency and go through the list search for peaks with identical coordinates. Double-click on them and adjust their position based on the 15N_HSQC.- At the end count the peak markers in both spectra using lt. They must be the same. If not, check for peaks with the same coordinates - they are seen as one.
• These peaks will be used as reference points for restricted peak picking in the 4D spectrum later on.

Below you see the final peak selection on the N-HN_proj (orange) adjusted to the peak centers. The 15N_HSQC is overlaid with cyan and yellow color. Notice that some peaks that were not present in the N-HN_proj (less sensitive experiment) were added from the 15N_HSQC and the inverse (although much fewer peaks were added like this).

#——————————————

Pick all peaks in the HC-C projection that match with reference 1H-13C HSQC
Since 1H-13C HSQC is very crowded and is not ideal for setting landmarks for restricted peak selection, we will use it in combinations ith the 2D_HC-C_proj. Overlay the two spectra and select with F8 all the peaks in 2D_HC-C_proj. The add manually the peaks that you believe are within the general boundaries defined by the 1H-13C HSQC and are not noise - a bit of intuition will be helpful here. Synchronizing the two spectra with yt and viewing them next to each other will help you. In the following Figure I show how I add a new peak in the 2D_HC-C_proj based on the density of the 1H-13C HSQC.

These peaks in 2D_HC-C_proj will be your landmarks for restrictive peak selection.

Optimizing Restricted Peak Picking for Higher Accuracy

• For more accurate restricted peak picking and to reduce noise, it is recommended to use different tolerances for peaks based on their radius.
• Open each projection spectrum, sort the peaks by data height, and start the lowest intensity peaks.
• Double-click on peaks in the lt table to visualize them, hit ms to measure their radius in both axes and group them based on suitable tolerance values that will capture all corresponding peaks in the 4D spectrum within that region of the 2D plane. In general applying the same tolerance to all peaks with similar data height is a good strategy.
• Once you decide about the tolerance values of a group of peaks, select them in the lt window and hit nt to save in a note the chosen tolerance values.

Since this protein is large, we will perform restricted peak picking in two rounds.
This is necessary because the screen updates every time a picking cycle completes, and for large proteins, this eventually becomes terribly slow.

• First, select approximately half of the peaks in the N-HN projection.
• Press kr to open the Restricted Peak Picking window. Make sure you copied our enhanced plugin to your POKY distribution (see Prerequisites)!
• Activate the toggle options:
  • Use selected peaks only
  • Use tolerance values from note
• Under Find peaks, select the 4D NOESY spectrum.
• Under Using peaks in, select the N-HN projection.
• Click the Pick Peaks button.
• Once it finishes, repeat restricted peak picking, this time:
  • Set Using peaks in to the HC-C projection
  • Deactivate the toggle Use selected peaks only
  • Click the Select Peaks button.
• Then, switch to the 4D NOESY window, press I (capital i), and then the Delete button to remove all irrelevant peaks.
• The remaining peak list displayed on the HC-C plane of the 4D NOESY should now look clean.
• Press pa (select all), then NT, and write a word like matched to mark these peaks.

• Click Apply.

• Now go back to the N-HN projection window.
• Select the remaining half of the peaks.
• Open the Restricted Peak Picking window again.
  • Set Using peaks in to the N-HN projection.
  • Activate both toggle options:
    • Use selected peaks only
    • Use tolerance values from note
  • Click the Pick Peaks button.
• Change Using peaks in to the HC-C projection.
• Deactivate Use selected peaks only.
• Click Select Peaks.
• Switch to the 4D NOESY window, press NT, write the word matched, and click Apply.

⚠️ Important: Do not delete any peaks yet, as we did in the first round—otherwise, you will lose some of the peaks identified earlier.

• Press lt to display the pick list in the 4D spectrum.
• Click Options, sort the list by Note, activate the Note toggle, and click Apply.
• Our goal is to delete only those peaks that do not have a note. The good peaks are those with the word matched in the Note column.
• Use the Page Up and Page Down keyboard buttons to scroll quickly and select large portions of peaks without notes.
• Once a significant portion is selected, switch to the 4D NOESY window and press the Delete key to remove the selected irrelevant peaks.

💡 For large proteins with tens of thousands of peaks, it is recommended to delete them in two batches rather than all at once.

This is how the final peak selection on the HC-C plane of the 4D NOESY should look.

[FIGURE]

Step X. Unalias/Unfold 4D Peaks

Next, we will perform unaliasing/unfolding of peaks. For more details, please read the respective article.

Aliased Peaks usually occur in the ranges C < 25 ppm and HC > 3 ppm.

In this spectrum, we have some aliased peaks that appear on top.

• Press F1 to switch to selection mode, select the aliased peaks, and then press a1 to unalias them along the C axis (W1).

[FIGURE here]

---

Step X. Manual Refinement of 4D Peak List

Next, we will manually inspect all the peaks and remove those that are not located in density regions—neither of the HC-C projection nor of the N-HN projection.

• Type fo and load the 4D spectrum again, this time to a new window.
• In the new window double-click xr followed by xx to bring the N and HN axes into view.
• Adjust the view by pressing vt and increasing the aspect.
• Press vz, set the values to:
  • N and HN axes = 0
  • C and HC axes = 9999
  • Click Apply.
• You might need to adjust the peak sizes by typing oz.
• Hit ol and overlay:
  • First the N-HN projection onto the 4D
  • Then the 15N HSQC onto the 4D
• Hit st and rename this window to 4D_HCNH_NOESY - N-HN proj

You should now have two different views of the 4D spectrum:

1. One showing the selected peaks on the HC-C plane
2. The other showing the selected peaks on the N-HN plane

What you must do next is manually inspect the peaks and delete those not in density regions. This requires a bit of intuition and a sharp eye. Unfortunately, it cannot be automated—it must be supervised manually by pressing st.

Discard the noise peaks using a S/N cutoff

• Hit st and in the text box “noise as median of” write 10000 or another high number.
• Click “Recompute” several times.
• If the “Estimated noise:” changes a lot, increase the “noise as median of” and repeat the process.
• Once you settle on an “Estimated noise:” value, open the peak list by hitting lt, display the S/N and sort by Data Height.
• Select all peaks with absolute S/N value less than the “Estimated noise:” and delete them. For stricter peak picking, you can set the cutoff to 3x or 5x the “Estimated noise:”.

** OPTIONAL: Discard the noise peaks using a weak peak as a reference**

• Overlay the N-HN_proj onto the 15N_HSQC and increase the contour levels until noise peaks start to emerge.
• Select a 15N_HSQC peak with weak signal in N-HN_proj and check its HC-C plane by switching to 4D_HCNH_NOESY and hitting vc.
• Review the picked peaks (previously found by kr), adjusting the contour levels until all visible peaks are captured.
• If you cannot achieve this, pick another weak peak in the 15N_HSQC and repeat the process.
• Once you succeed, select the weakest picked peak and hit ss. This command will select all 4D peaks that have lower intensity than the selected one. Type lt to check that.
• Click Delete to remove them.

Note: This comparison is made using signed intensities: negative peaks with large absolute intensity will also be selected! Check them manually and deselect if needed: hold Ctrl and drag over the selected peaks with your mouse.

(^ There should be a GIF here showing large negative peaks.)

---

Step X. Exporting Peak Lists

Export Picked Peaks for 4D-GRAPHS
Go to the 4D peak list (type lt) and select the columns w1, w2, w3, w4, Data Height and Note. Click Apply, then Save…. Further editing of the projections and the HSQCs is needed.

Improve the Precision of the 13C HSQC Peak List using the 4D HCNH NOESY Peak List

• Once you have finalized the peak list from the HC-C projection, export it to a file.
• Press fo and reopen the 13C HSQC spectrum in a new window.
• Press st and rename that window to “13C HSQC - 4D Peak List”.
• Hit rp, toggle on Auto detect dimensions and load the 4D HCNH NOESY peak list.
• Verify that the aliased/folded peaks are unaliased/unfolded, just as on the 4D spectrum (POKY does this automatically).
• Hit lt and through the “Options” display the “Data Height”.
• Export the updated peak list to a new file named 13C_HSQC_with_4D_HCNH_NOESY_peaks.list.

We follow this approach because the 13C HSQC spectrum is very noisy, with large dispersion effects, meaning that the peak centers deviate from those identified in 4D HCNH NOESY spectrum.
Consequently, since the entire assignment relies on the 4D spectrum, it is more accurate to use the peak markers from it, but enhanced with the intensity signs present in the 13C HSQC spectrum.
The 13C HSQC spectrum provides information on whether a peak corresponds to a methylene group, which improves both accuracy and coverage for chemical shift assignment in 4D-GRAPHS.

Improve the Precision of the 15N HSQC Peak List using the 2D N-HN projection Peak List

The 15N HSQC peak list for 4D-GRAPHS must have only one peak for each spin system, therefore we will apply the previous trick but using the `*N-HN projection peak list instead of the 4D HCNH NOESY peak list.

---

Notes for Special Cases

Unaliasing Peaks in POKY
When you do restricted peak picking (kr) using as a reference Peaks that have not been unaliased or unfolded, POKY will automatically check for possible aliased peaks. If the spectrum width of the source 2D is larger than that of the nD (n = [3,4]), POKY will find and mark the peaks in the 3D as aliased.

However, BEWARE that when your reference peaks are aliased or unfolded, POKY won’t match the correct peaks in the target spectrum unless they are also unalias/unafolded. It may catch some peaks but they will be irrelevant. Therefore, do not unalias/unfold the peaks in the 2D HC-C and N-HN projections! Do the unaliasing/unfolding directly on the 4D HCNH NOESY.

Below are examples of the 13C-HSQC spectra with aliased peaks (in yellow boxes):

Protein 1	Example 13C-HSQC - Protein 2

---

Authors

• Thomas Evangelidis
• Ekaterina Burakova