Feb 19, 2025

Public workspaceBio - hCONH 3D

DOI
dx.doi.org/10.17504/protocols.io.4r3l2q28jl1y/v1
  • Songlin Wang1
  • 1NMRFAM, University of Wisconsin-Madison
NMRFAM Facility
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2q28jl1y/v1
Protocol Citation: Songlin Wang 2025. Bio - hCONH 3D. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q28jl1y/v1
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: 100291
Keywords: 3D hCONH
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose 
hCONH 3D heteronuclear dipolar correlation 1H detection experiment at fast spinning rate.  

Scope 
Intra-residue (COi-1, Ni, Hi) backbone chemical shift correlations of CO, N and H atoms. 3D hCONH is used for sequential assignment of 15N, 13C, and 1H chemical shifts in conjugation with 3D hCANH and 3D hCA(CO)NH experiments. 

Checklist for setting hCONH3d experiment:
  1. Load parameters file or previous standard experiment acquired using the same spectrometer, probe, and spinning rate (if exist).
  2. Updated the optimized parameters (p14, plw14, p13, plw13, p15, plw15, p43, spnam41, cnst41, spnam31, cnst31, p35, spnam32, cnst32, spnam52, cnst52, p54, spnam53, cnst53, spnam43, cnst43, p38, spnam38, cnst38, cpdprg4, p24, and cnst24, cpdprg3, p23, cnst23, cpdprg5, p25, cnst25, cpdprg6, p26, cnst26, and d30).
  3. Set carrier frequencies for each channel (O1p, O2p, O3p).
  4. Set acquisition time (aq), dwell time (dw), spectral width (sw), and complex points (TD) for each dimension.
  5. Set recycle delay (d1)
  6. Set NUS schedule if applicable
  7. Set scan numbers (NS)
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:
TermDefinition
hCANHCP based HNCA 3D correlation experiment
CPCross Polarization
MASMagic Angle Spinning
NUSNon-Uniform Sampling
Instrument: The hCONH3d example dataset in this SOP was acquired using a 600 MHz NMR spectrometer with Bruker Avance III HD console. The probe used was a Phoenix 1.6mm HCN probe. For the best result, this experiment should be executed after properly adjusting the shimming and magic angle.

Sample:
  1. Type: Protein
  2. Labeling: Perdeuterated and 1H back exchanged U-13C,15N, 2H labeled samples (default) or fully protonated U-13C,15N labeling samples at spinning speed higher than 60 kHz.
  3. Minimum amount: ~50 nmol for 1.6 mm rotor

hCONH3d schematic pulse sequence:




Example parameter set for a Phoenix 1.6mm probe using 38.462 kHz MAS on 600 MHz Bruker spectrometer (pulse sequence hCNH3d-NAN). Sample is 25% 1H back exchanged, 15N, 13C, 2H -GB1 protein:

Pulse Parameters:
1H 90o: (p14, cnst14) = (2.5 μs, 100 kHz)
13C 90o: (p13, cnst13) = (2.5 μs, 100 kHz)
15N 90o: (p15, cnst15) = (5 μs, 50 kHz)
HCA CP: [p43, cnst41(1H), cnst31(13C)] = [5.0 ms, 77 kHz (tan70to100up.1000), 27 kHz (cw)]

CAN-CP: [p35, cnst32 (13C), cnst52 (15N)] = [10.0 ms, 21 kHz (cw), 19 kHz (tan70to100up.1000)]

NH-CP: [p54, cnst43 (1H), cnst53 (15N)] = [1.0 ms, 54.5 kHz (tan70to100up.1000), 11 kHz (cw)]

CA selective pulse: [p38, cnst38 (RSNOB)] = [273 µs, 9 kHz]

­1H-decoupling - (CPDPRG4, p24, cnst24) = (SPINAL64_p24, 184 μs, 2.5 kHz)

­­13C-decoupling - (CPDPRG3, p23, cnst23) = (WALTZ16_p23, 50 μs, 10 kHz)

­­15N-decoupling - (CPDPRG5, p25, pl25) = (WALTZ16_p25, 50 μs, 10 kHz)
1H-solvent suppression - (CPDPRG6, p26, cnst26, d30) = (MISSISSIPI_p26, 50 ms, 10 kHz, 0.2s)

Acquisition Parameters:
MAS rate (wr) in Hz: cnst20 = 38.462 kHz
Rotor period (tr): d20 = (1/cnst20) = 26.0 s
1H carrier frequency: O1p = 6 ppm
Number of scans: ns = 4
Recycle delay: d1 = 1 sec

1H Direct dimension: t3 or F3:
Carrier frequency: O1p = 6 ppm
Acquisition time: aq(F3) = 28 ms
Spectral width: sw(F3) = 30.5 ppm

CA indirect dimension: t1 or F1:
Carrier frequency: O2p = 175 ppm
Maximum evolution time: aq (F1) = 18.3 ms
Spectral width: sw (F1) = 19.6 ppm
Total t1 points: TD (F1) = 108
Complex t1 points: 54

15N indirect dimension: t2 or F2:
Carrier frequency: O3p = 118 ppm
Maximum evolution time: aq (F2) = 13.1 ms
Spectral width: sw (F2) = 35.2 ppm
Total t2 points: TD (F2) = 56
Complex t2 points: 28
Safety warnings
Operator: User should be knowledgeable to operate MAS probes and use Topspin. User should know the power limits of the MAS probe being used.
Before start
MAS rate: ~40 kHz for 1.6 mm rotor or ~60 kHz for 1.2 mm rotor.

Temperature: As determined for optimal sample sensitivity and resolution.

Below optimizations are required before setting up the hCONH3d 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).
  1. Calibration of 1H, 13C, and 15N solid pulses (p14, plw14, p13, plw13, p15, and plw15)
  2. Optimization of HCO CP (p43, spnam41, spnam31, cnst41, and cnst31)
  3. Optimization of CON CP (p35, spnam32, spnam52, cnst32, and cnst52)
  4. Optimization of NH CP (p54, spnam53, spnam43, cnst53, and cnst43)
  5. Optimization of CO selective pulse (p38, spnam38, cnst38)
  6. Optimization of low-power 1H decoupling (cpdprg4, p24, and cnst24)
  7. Optimization of low-power 13C decoupling (cpdprg3, p23, and cnst23)
  8. Optimization of low-power 15N decoupling (cpdprg5, p25, and cnst25)
  9. Optimization of 1H solvent suppression pulse (cpdprg6, p26, cnst26, and d30)

Setup time: ~15 min, presuming above optimizations have already been completed.
Procedure
Procedure
Load “hCNH3d-NAN” pulse program and parameter set “hCONH3d-NAN_par”.
Use Topspin command “edc” to open a new experiment.
Input “hCNH3d-NAN” as PULPROG
Type “rpar”, then load file “hCONH3d-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 ~5ppm (for the best water suppression performance). Set the 13C carrier frequency at ~175 ppm, and 15N carrier frequency at ~ 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 (t3 or F3)
  1. 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
  2. Default spectral width: ~30 ppm.
Indirect 13C dimension (t1 or F1)
  1. Increment of delay (IN_F, s): n * tr. For example, 182 μs can be used for 38.462 kHz MAS on 600 MHz. For a given MAS rate, adjust the dwell time to integer multiples of tr that covers at least 20 ppm 13C spectral width.
  2. Spectral width (SW, ppm): coupled with dwell time, >20 ppm is required to cover the full spectrum.
  3. Carrier frequency (O2p, ppm): 175 ppm.
  4. Hypercomplex scheme (FnMODE): States-TPPI
  5. Maximum evolution time (AQ, s): depending on 13CO T2, the default is ~10 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.
Indirect 15N dimension (t2 or F2)
  1. Increment of delay (IN_F, s): n * tr. For example, 468 μs can be used for 38.462 kHz MAS on 600 MHz. For a given MAS rate, adjust the dwell time to integer multiples of tr that covers at least 40 ppm 15N spectral width.
  2. Spectral width (SW, ppm): coupled with dwell time, >30 ppm is required to cover the full spectrum.
  3. Carrier frequency (O3p, ppm): 118 ppm.
  4. Hypercomplex scheme (FnMODE): States-TPPI
  5. Maximum evolution time (AQ, s): depending on 15N T2, the default is ~10 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 for maximum sensitivity per unit time. If 1H T1 is not measured, use the default value which is 2 s. However, measuring 1H T1 before setting this experiment is highly recommended for best practice.
Set NUS schedule. This step is optional, but 25% NUS is recommended for 3D experiments
Adjust measurement time as required by increasing number of scans in multiples of 16.
Validation
Start the experiment and monitor the first ~20-30 rows.
Process first dimension FT to check for adequate signal correctly arraying indirect dimension.
Protocol references