Feb 19, 2025

Public workspaceBio - hNCACX 3D

This protocol is a draft, published without a DOI.
  • Songlin Wang1
  • 1NMRFAM, University of Wisconsin-Madison
NMRFAM Facility
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Protocol Citation: Songlin Wang 2025. Bio - hNCACX 3D. protocols.io https://protocols.io/view/bio-hncacx-3d-dd6829hw
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: 100288
Keywords: 3D hNCACX
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose 
Three-dimensional hNCACX dipolar correlation experiment with CC DARR mixing at moderate spinning rate.  

Scope 
Intra-residue (Ni,CAi,CXi) backbone and sidechain chemical shift correlations of N, CA and CX atoms using DARR mixing times of 25 to 50 ms. Inter-residue correlations with moderate DARR mixing times of 50-200 ms. 3D hNCACX is used for sequential assignment of 15N and 13C chemical shifts in conjugation with 3D hNCOCX and/or hCANCO. 

Checklist for setting hNCACX3d 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, p45, spnam41, cnst41, spnam51, cnst51, p53, spnam32, cnst32, spnam52, cnst52, p38, spnam38, cnst38, cpdprg4, p24, and cnst24).
  3. Set carrier frequencies for each channel (o1p, o2p, o3p).
  4. Set desired DARR rf-power and mixing time (cnst5, d5)
  5. Set acquisition time (aq), dwell time (dw), spectral width (sw), and complex points (TD) for each dimension.
  6. Set recycle delay (d1)
  7. Set NUS schedule if applicable
  8. 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 at section 6.10 to find corresponding parameter names if using other pulse sequences.  
Materials
Definitions:
TermDefinition
hNCACXCP based NCACX 3D correlation experiment
DARRDipolar assisted rotational resonance
MASMagic angle spinning
CPCross polarization
NUSNon-Uniform sampling
Instrument: The hNCACX3d example dataset in this SOP was acquired using a 600 MHz NMR spectrometer with Bruker Avance III console. The probe is a Phoenix 1.6 mm HCN probe. For the best results, this experiment should be executed after properly adjusting the shimming and magic angle.

Sample:
  1. Type: Protein
  2. Labeling: Fully protonated U-13C,15N labeling (default), or 13C-glycerol skip-labeled (alternative conditions where noted). Not applicable to perdeuterated samples.
  3. Minimum amount: ~ 50 nmol for 1.6 mm rotor

hNCACX3d schematic pulse sequence



Example Parameter set for a Phoenix 1.6 mm probe using 13.333 kHz MAS on 600 MHz Bruker spectrometer (pulse sequence: NCC3d-NAN)

Pulse Parameters:
1H 90o: (p14, plw14, cnst14) = (2.5 ms, 43W, 100 kHz)
13C 90o: (p13, plw13, cnst13) = (2.5 ms, 150W, 100 kHz)
15N 90o: (p15, plw15, cnst15) = (5 ms, 125W, 50 kHz)
HN CP: (p45, spnam41, cnst41, spnam51, cnst51) = (1.8 ms, tan70to100, 79 kHz, cw, 33.33 kHz)
NCA CP: (p53, cnst42, spnam32, cnst32, spnam52, cnst52) = (7.5 ms, 100 kHz, tan70to100, 21 kHz, cw, 33.33 kHz)
CA selective pulse: (p38, spnam38, cnst38) = (262.5 ms, RSNOB, 8.2 kHz)

DARR mixing: (d5, cnst5) = (50 ms, 13.333 kHz)

­­1H-decoupling - (CPDPRG4, p24, cnst24) = (SPINAL64_p24, 5.2 ms, 90 kHz).

Acquisition Parameters:
MAS rate (wr) in Hz: cnst20 =13333
Rotor period (tr): d20 = (1/cnst20) =75 ms
1H carrier frequency: O2p = 10 ppm
Number of scans: ns =16
Recycle delay: d1 = 1.0 sec
13C Direct dimension: t3 or F3:
Carrier frequency: O1p = 55 ppm
Acquisition time: aq(F3) = 13.6 ms
Spectral width: sw(F3) = 497.278 ppm
15N indirect dimension: t1 or F1:
Carrier frequency: O3p = 118 ppm
FnMODE: States-TPPI
Maximum evolution time: aq(F1) = 10.5 ms
Spectral width: sw(F1) = 43.9 ppm
Complex t1 points: TD (F1) = 56
13CA indirect dimension: t2 or F2:
Carrier frequency: 55 ppm
FnMODE: States-TPPI
Maximum evolution time: aq(F1) = 7.2 ms
Spectral width: sw(F2) = 29.5 ppm
Complex t2 points: TD (F2) = 64

Safety warnings
Operator: User should be knowledgeable to operate MAS probes and use Topspin. User should know the power limit of the MAS probe being used.
Before start
MAS rate: 13.333 kHz at 600 MHz, 16.667 kHz at 750 MHz, or 20 kHz at 900 MHz (~88.9 ppm in 13C). Note that the transfer efficiency of DARR decreases significantly when MAS > 30 kHz

Temperature: As determined for optimal sample sensitivity and resolution.

Below optimizations are required before setting up the hNCACX3d 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 HN CP (p45, spnam41, cnst41, spnam51, and cnst51)
  3. Optimization of NCA CP (p53, spnam32, cnst32, spnam52, and cnst52)
  4. Optimization of CA selective pulse (p38, spnam38, cnst38)
  5. Optimization of high-power 1H decoupling (cpdprg4, p24, and cnst24)

Setup time: ~15 min, presuming above optimizations have already been completed.
Procedure
Procedure
Load pulse program and parameter set. “NCC3d-NAN” pulse program and parameter set “hNCACX3d-NAN_par”.
If a previous hNCACX3d experiment acquired using the same setup is available (i.e., same spectrometer, probe, pulse program and MAS rate), open the previous dataset and type “edc”. Input the directory for the new experiment and press “OK”.
If no previous data available, then use Topspin commend “edc” to open a new experiment. Input “NCC3d-NAN” as PULPROG. Type “rpar”, then load “hNCACX3d-NAN_par”.
Set the 1H carrier frequency (O2p) at ~5 ppm, 13C carrier frequency (O1p) at 55 ppm, and 15N carrier frequency (O3p) at 118 ppm. For example, type “O1p” and enter, then input 55 to the opened window.
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 DARR mixing. Set the DARR mixing.
Set the DARR 1H condition. The 1H rf power for DARR mixing should match the MAS rate. For the pulse sequence shown at "Materials", the 1H rf power for DARR mixing is cnst5. The unit of cnst5 is kHz.
Choose DARR mixing time
  1. For short range intra-residue correlations, set the DARR mixing to 10-50 ms which is optimal for sequential correlation of 2D planes along with other 3D data sets (i.e., hNCOCX3d and hCANCO3d).
  2. For medium range inter-residue correlations set the DARR mixing to 50-200 ms.
  3. For long range (>5 Å) correlations set the DARR mixing to >200 ms.
  4. The parameter name of DARR mixing time is d5 and the default unit is s. For example, if 50 ms is used, type “d5” and input 0.05. Optimal mixing times increase with magnetic field.
Set the acquisition parameters.
Direct dimension (t3 or F3)
  1. Default acquisition time: depending on 13C T2, default is ~20 ms (alternative for 13C-glycerol: ~30 ms).
  2. Default spectral width: ~300 ppm.
  3. Default carrier frequency: 55 ppm.
Indirect 15N dimension (t1 or F1)
  1. Increment of delay (IN_F, s): n * τr. For example, 375 ms can be used for 13.33 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, >40 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.
Indirect 13C dimension (t2 or F2)
  1. Increment of delay (IN_F, s): n * τr. For a given MAS rate, adjust the dwell time to integer multiples of τr that covers at least 30 ppm 13C spectral width.
  2. Spectral width (SW, ppm): coupled with dwell time, >30 ppm is required to cover the full spectrum
  3. Carrier frequency (O1p, ppm): 55 ppm.
  4. Hypercomplex scheme (FnMODE): States-TPPI
  5. Maximum evolution time (AQ, s): depending on 13C 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.
Due to high-power 1H decoupling is applied, don’t set recycle delay less than 1s.
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
Reference: J. Pauli, M. Baldus, B. van Rossum, H. de Groot, H. Oschkinat, Backbone and side-chain 13C and 15N signal assignments of the aspectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla, ChemBioChem 2 (2001) 272–281. https://doi.org/10.1002/1439-7633(20010401)2:4<272::AID-CBIC272>3.0.CO;2-2

SOP: 2.2.1.1 1H/13C/15N 90o pulse width calibration

SOP: 2.2.1.2.1 1H-13C/1H-15N Cross Polarization

SOP: 2.2.1.3.1.1 1H high-power decoupling

SOP: 2.2.2.1.1.7.1 15N-13C specific CP

SOP 2.2.1.1.2.1.1 13C selective pulse optimization

SOP 2.2.2.1.1.1.1 1H T1 Measurement

SOP 2.2.2.1.1.2.2.1 13C T2 Measurement