Feb 19, 2025
Bio - hNH 2D
This protocol is a draft, published without a DOI.
- Songlin Wang1
- 1NMRFAM, University of Wisconsin-Madison
Protocol Citation: Songlin Wang 2025. Bio - hNH 2D. protocols.io https://protocols.io/view/bio-hnh-2d-dd6s29ee
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 22, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 100274
Keywords: 2D hNH
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose
Two-dimensional hNH dipolar correlation 1H detection experiment at fast spinning rate.
Scope
Sample quality assessment and protein NMR fingerprint.
Guidelines
This SOP is written based on the pulse sequence developed at NMRFAM. If using other pulse sequences, the strategy is still applicable, but the parameter names will be different. Please refer to the schematic pulse sequence under "Materials" to find corresponding parameter names if using other pulse sequences.
Materials
Definitions:
| Term | Definition | |
| hNH | CP based HN 2D correlation experiment | |
| CP | Cross Polarization | |
| MAS | Magic Angle Spinning |
Instrument: The hNH2d experiment described in this SOP was acquired using a 600MHz NMR spectrometer with a Bruker Avance III console. The probe is a Phoenix 1.6 mm HCN probe. For the best result, this experiment should be executed after properly adjusting the shimming and magic angle.
Sample:
- Type: Protein
- Labeling: perdeuterated U-15N labeling with 1H back exchanged samples.
- Minimum amount: ~ 50 nmol for 1.6 mm rotor
hNH2d schematic pulse sequence:
Example parameter set tested on protein sample:
Spectrometer: Fleckvieh (900 MHz) at NMRFAM
Probe: Phoenix 1.6 mm HCN probe
Spinning: 38.462 kHz
Sample: 25% 1H back exchanged, 15N, 13C, 2H -GB1
Pulse Parameters:
1H 90°: (p14, cnst14) = (2.5 µs, 100 kHz)
13C 90°: (p13, cnst13) = (5 µs, 50 kHz)
15N 90°: (p15, cnst15) = (9.6 µs, 26.0 kHz)
HN CP: [p45, cnst41(1H), cnst51(15N)]
= [2.50 ms, 35.5 kHz (tan70to100), 10 kHz (cw)]
NH-CP: [p54, sp42 (1H), sp52 (15N)]
= [1.25 ms, 31 kHz (tan70to100), 10 kHz (cw)]
1H-decoupling - (CPDPRG4, p24, cnst24)
= (SPINAL64_p24, 46 µs, 10 kHz)
13C-decoupling - (CPDPRG3, p23, cnst23)
= (WALTZ16_p23, 25 µs, 10 kHz)
15N-decoupling - (CPDPRG5, p25, cnst25)
= (WALTZ16_p25, 25 µs, 10 kHz)
1H-solvent suppression - (CPDPRG6, p26, cnst26, d30)
= (MISSISSIPPI_p26, 50 ms, 30 kHz, 0.2 s)
Acquisition Parameters:
MAS rate (wr) in Hz: cnst20 = 38462
Rotor period (tr):
d20 = (1/cnst20) = 26 µs
Number of scans: ns = 4
Recycle delay: d1 = 1 sec
1H Direct dimension: t2 or F2:
Carrier frequency: O1p = 11 ppm
Acquisition time: aq(F2) = 39.9 ms
Spectral width: sw(F2) = 42.7 ppm
15N indirect dimension: t1or F1:
Carrier frequency: O3p = 120 ppm
Maximum evolution time: aq(F1) = 41.6 ms
Spectral width: sw(F1) = 42.181 ppm
Dwell time: IN_F (F1) = 260 µs
Total t1 points: TD (F1) = 160
Complex t1 points: 320
Safety warnings
Operator: User should be knowledgeable to operate MAS probes and use Topspin
Before start
MAS rate: 35-40 kHz for 1.6 mm rotor
Temperature: Note that the temperature increases by 15 -20 °C due to frictional heating from spinning at 35-40 kHz for 1.6 mm rotors. Adjust the variable temperature (VT) control set point temperature according to
achieve desired sample temperature. For microcrystals and fibrils, typical sample temperature range is 0 to 10 °C, requiring a VT set point temperature of -20 to -5 °C. For membrane proteins and/or liposomes, the desired sample temperature depends on the phase transition of interest and can vary over a wide range depending on
the sample.
Below optimizations are required before setting up the hNH2d experiment (parameters needs to be updated are shown in parenthesis). Example SOP for each optimization is attached at References. Note that RF powers in this pulse sequence defined using kHz rather than Watt (i.e., input kHz number for rf powers and the code will calculate the corresponding Watt numbers for Topspin to use). Only the hard pulses for calibration need to be input as Watt (plw13, plw14, and plw15).
- Calibration of 1H, 13C, and 15N solid pulses (p14, plw14, p13, plw13, p15, and plw15)
- Optimization of HN CP (p45, spnam41, spnam51, cnst41, and cnst51)
- Optimization of NH CP (p54, spnam52, spnam42, cnst52, and cnst42)
- Optimization of low-power 1H decoupling (cpdprg4, p24, and cnst24)
- Optimization of low-power 13C decoupling (cpdprg3, p23, and cnst23)
- Optimization of low-power 15N decoupling (cpdprg5, p25, and cnst25)
- Optimization of 1H solvent suppression pulse (cpdprg6, p26, cnst26, and d30)
Setup time: ~0.5 hours, presuming initial calibrations have already been completed.
Procedure
Procedure
Load “hNH2d-NAN” pulse program and parameter set “hNH2d-NAN_par”.
Use Topspin command “edc” to open a new experiment.
Input “hNH2d-NAN” as PULPROG
Type “rpar”, then load file “hNH2d-NAN_par”.
Note that the parameters will be off if using different spectrometers/probes. Please consult to your facility manager/staff to get proper starting parameter set.
Set the 1H carrier frequency at ~5 ppm (for the best water suppression performance). Set the 13C
carrier frequency at ~100 ppm, and 15N carrier frequency ~ 118 ppm.
Optimize the pulse sequence parameters. The optimization needs to be done are listed in the "Before start" section under "Guidelines & Warnings". Example SOPs for the required optimization are attached at "References". Note that the parameters can be optimized use different methods/pulse sequences. Please consult to your facility manager to perform the optimization.
Set the acquisition parameters.
Direct 1H dimension (t2 or F2)
- Default acquisition time: ~50 ms. Note that the acquisition time depends on the 1H T2. However, the low power decoupling allows us use longer acquisition time. So, we recommend acquiring ~50 ms if the 1H T2 is unknown. The FID can be truncated using data processing software (Topspin or NMRPipe) readily
- Default spectral width: ~30 ppm.
Indirect 15N dimension (t1 or F1)
- Increment of delay (IN_F, s): n * tr. For a given MAS rate, adjust the dwell time to integer multiples of tr that covers at least 40 ppm 15N spectral width. For example, 468 μs can be used for 38.462 kHz MAS on 600 MHz.
- Spectral width (SW, ppm): ~ 200 ppm. Note that the 15N spectral width only need to cover the chemical shift range of amide 15N which is typically from 100 to 140 ppm. However, if the spectral pattern is unknown, we recommend using ~200 ppm to prevent any side-chain resonances from folding back into amide region. Adjust "Increment of delay" to change SW.
- Carrier frequency (O3p, ppm): 118 ppm.
- Hypercomplex scheme (FnMODE): States-TPPI
- Maximum evolution time (AQ, s): depending on 15N T2, the default is ~20 ms. The evolution time equals to (Increment of delay) * (TD). Once increment of delay is fixed as described above, change number of TD to adjust the total evolution time.
Set the recycle delay.
Use recycle delay of 1.3*T1(1H) for maximum sensitivity per unit time. If 1H T1 is unknown, use 2 s. 1H T1 measurement is recommended if it is unknown.
Determine experiment time
Minimal number of scans (phase cycle limited): 4
Minimal measurement time: ~10 min
Adjust measurement time as required for sample by incrementing number of scans in multiples of 4.
Validation
Start the experiment and monitor the first ~20-30 rows.
Process first dimension FT (xf2) to check for adequate signal correctly arraying indirect dimension.
Protocol references