OBSOLETE!!! Guide to Set Up 4D NMR Experiments

Setting Up NMR Experiments

Downloading Ubiquitin’s Parameter Set

For a smooth configuration of your spectrometer, download Ubiquitin’s parameter set (the Topsin directory including spectra and parameters) from this Google drive link. This parameter set is from a 600 MHz magnet (e.g. generated with “wpar”) and can be adapted to your machine using “paracon” and some further fiddling.

In the “Complete_experiments” folder, you will find the following experiments:

Next, you can find experiments for individual planes of the 4D experiment derived from Experiment 101:

In the “WPAR_all” directory, you will find parameter sets from all experiments, as these were generated for each one. They are from our 600 MHz spectrometer because the most recent experiments were conducted on it, and I have the processing pipeline set up for those pulse programs.

Download the planes from 4D HCNH NOESY measured on our 850 MHz with Ubiquitin sample. You can use the data either to check the parameters or do the setup from our parameters directly. If you chose the other option, you definitely have to go first to edasp and click default to set wiring and frequencies for your hardware. Also, you may need to change DIGTYP to the one your hardware uses. Then you need to recalibrate the pulses and that should be it. I suggest not to use getprosol as the pulses may be used in a different way than Bruker assumes. It is important to check first with an easy sample that you are getting signals, and the spectrum looks as expected.

Google Drive Link

Feasibility Assessment with 2D Experiments

15N HSQC (hsqcedetf3gpsi2)

Begin with the 15N HSQC using the hsqcedetf3gpsi2 pulse sequence to obtain in-phase peaks for N-H2 and anti-phase peaks for side-chains N-H. This experiment is necessary for chemical shift assignment with 4D-GRAPHS.

13C HSQC (hsqcedetgp)

Conduct a 13C HSQC experiment for in-phase peaks of CH, CH3, and anti-phase for CH2. This experiment is also necessary for chemical shift assignment with 4D-GRAPHS. We don’t measure the aromatics in the 13C HSQC. The reason is that we use 13C HSQC hsqcedetgp just to identify the CH2 for the assignment. Besides that, the 4D HCNH NOESY is primarily for the aliphatic C-H - we see only part of the aromatic carbons in the NOESY. But in any case, it’s useful to have the aromatic C-H in 13C HSQC, but for them, we don’t need the hsqcedetgp.

Quality Assessment

Evaluate the HN, HC planes of 4D HCNH NOESY to assess quality indicators such as signal-to-noise ratio, peak sharpness, cross-peak intensity, and the absence of artifacts. Additionally, check the H-H 2D plane for calibrating the mixing time for the 4D NOESY experiment(s). We frequently use intermediate mixing time (e.g. 70 ms).

NUS Setup

We use Topspins’s NUS schedule generator. The number of NUS points and % sampling will depend on you sample. Specifically, on the required number of scans, D1 delay, and other parameter values required to obtain good S/N. the available spectrometer time.

Measurement Setup for HSP90 (25 kDa) on an 850 MHz Spectrometer

Goal: prepare and launch multiple 2D planes from various 4D HCNH NOESY pulse programs, evaluate their quality, and choose the most suitable 4D sequence.

Chronological Command Log

Command Purpose / Action
1s te Set the temperature.
lock Opens a solvent‐selection dialog, reads lock parameters (from edlock table), and performs autolock. Suitable for solvents with multiple lock signals. Selected solvent: 90 % H₂O / 10 % D₂O.
edasp (opens ASP editor; parameters not modified in this run).
p1, p3, p21 Display current pulse lengths.
atma Automatic tuning & matching for every nucleus; repeat for every sample and probe.
atmm Manual fine-tuning after atma. Adjust two parameters:
 1. Tuning – align probe resonance with Larmor frequency.
 2. Matching – match probe impedance (50 Ω). Warning: correcting one nucleus may disturb others.
topshim gui Automatic / manual shimming (homogenize B₀ in z and xy).
loopadj Optimize lock parameters LGAIN, LTIME, LFILTER.
pulsecalc Calibrate pulse lengths; essential for ¹H (¹³C/¹⁵N vary less).
getprosol … Retrieve / set pre-calibrated 90° hard-pulse parameters.
Example:
getprosol 1H 13.29 14.529W 13C 12.0 162.93W 15N 37.8 169.82W
O1 Set spectral center (in the observed dimension).
O1P, O2P, O3P Set transmitter-offsets (ppm) for 1st, 2nd, 3rd nuclei.
D1–D8 Delay parameters (s).
ZGOPTNS Add compiler flags (C-style -D<FLAG>) passed verbatim to the pulse programme.

Common ZGOPTNS Flags

Flag Typical pulse sets Enables Practical effect
-DLABEL_CN Triple-resonance (HNCA, HNCACB, HSQC, …) Double-labelled (¹³C/¹⁵N) branch Adds ¹³C decoupling during ¹⁵N evolution; un-comments P22, PL2, delays that depend on ¹JCN.
-DCALC_SP BEST / “b_…” selective or BEST-TROSY Auto-derives ¹H band-selective shapes from cnst54/55 Sequence generates & loads shaped pulses on the fly.

Other useful flags:

Flag Purpose
-DPRESAT Use continuous-wave presaturation during d1.
-DWG or -DWATERGATE Use WATERGATE suppression instead of presat.

Running the 2D Plane Experiments

  1. Iterate through every 2D plane you have set up.
  2. Launch each with zg (queued automatically).
  3. Inspect the resulting spectra; the best plane(s) will dictate the final 4D pulse program for this sample.

Setting Up the 4D HCNH NOESY

After selecting the optimal 2D planes, create the 4D HCNH NOESY experiment and copy these parameter values from the chosen 2D datasets:

Parameter Meaning
SW{Fx} Spectral width for each dimension F₁–F₄
IN_F{s} Increment for delay (µs)
CNST{2,4,16,18,19}{Fx} Various constants (mainly for ¹H; possibly for ¹³C and ¹⁵N)
SPNAM26{F4} / SPNAM1{F4} Shape file name for shaped pulse
P1{F4} / P17{F4} Length of the shaped pulse

Then run getprosol again with the updated 90° parameters, e.g.:

getprosol 1H 13.29 14.529W 13C 12.0 162.93W 15N 37.8 169.82W

Miscellaneous Commands

Command Description
ased Edit acquisition parameters used by the pulse program.
qfp Macro: applies a quadrature sine-bell window (qsin), performs FT, then phase correction (fp).

Notes & Practical Tips

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