Referencing Spectra in Topspin with BioTop (RECOMMENDED)

If you don’t have the BioTop Topspin extension, install the spectrometer-related dependencies by running expinstall and keeping the default values.
Execute biotop to open the browser window.
Set the measurement conditions correctly: Labeling, Solvent, Tube, Temperature. You can also provide the FASTA sequence of the protein, and BioTop will automatically calculate the Molecular size. (However, the MW does not affect the axes shift during referencing.)
Once ready, click Close and execute the command:
```
btproc biorefonly
```
You are all set. The axes should have shifted—check that they are correct.

Note
BioTop fails to reference 4D spectra and their 2D projections. In that case, you will use the 2D projections to align the 4D spectrum to the HSQC spectrum(s) during peak picking in POKY.

Calibrating (aka Referencing) Manually All Your 2D, 3D, 4D Spectra in Topspin

Calibration is the process of using a referenced spectrum (e.g., 1H-15N HSQC) to reference another spectrum (e.g., 4D HCNH NOESY).

Select the common axes between your 2D reference spectrum (for example, the 15N HSQC) and your 3D or 4D spectra.
- Example: For a 15N-edited 3D NOESY → F1: H, F2: N, F3: HN
  and a 4D HCNH NOESY → F1: C, F2: HC, F3: N, F4: HN
  you might choose F2-F3 (for the 3D) and F3-F4 (for the 4D) to match the dimensions of the reference 15N HSQC.
Create the N-HN positive projections to identify common peaks with strong signals (which will be used for calibration):
- For the 3D NOESY (15N-edited):
```
projplp 23 all all 23
```
- For the 4D HCNH NOESY:
```
projplp 34 all all 34
```
  This will create projections of the planes that correspond to the dimensions matching the 15N HSQC.
Pick a reference peak in the 15N HSQC:
- Go to the 15N HSQC, type .pp to manually pick a peak, select the chosen reference peak, type peaks, and note down its coordinates in ppm.
Go to the 3D spectrum and display its F2-F3 plane:
- Click on the “23” icon to display the F2-F3 plane.
- Type .md and select and drag the 15N HSQC spectrum into the display. In the left lower panel, increase contour levels to see the strong signals clearly without showing too much noise.
- Select the 3D spectrum and start navigating through the F1 axis by left-clicking and dragging on the “up-down arrow” icon.
- While you navigate, watch for the reference peak from the 15N HSQC that should start appearing as a strong signal in the overlaid F2-F3 plane.
- Once you see the strong signal of the selected reference peak, stop dragging, place your cursor at the peak center, and note its index(F1) coordinate (not the ppm coordinate).
Return to the F2-F3 plane in the 3D NOESY:
- Click the “return” icon to go back to the full F2-F3 plane view.
- Click the “E” icon and select the F2-F3 visible plane.
- Enter the index(F1) you noted down into the text box next to that plane.
Zoom in on the reference peak:
- You should now see the reference peak with a strong signal.
- Zoom in and type .cal, then left-click at the center of the reference peak.
- In the pop-up window, enter the ppm coordinates you noted from the 15N HSQC.
- Click OK. Both the F2 and F3 axes of the 3D NOESY will be shifted accordingly.
Repeat the procedure for the 34 projection of the 4D NOESY to calibrate it.

Authors

Thomas Evangelidis