Feb 20, 2025
Bio - CN-REDOR
This protocol is a draft, published without a DOI.
- Songlin Wang1
- 1NMRFAM, University of Wisconsin-Madison
Protocol Citation: Songlin Wang 2025. Bio - CN-REDOR. protocols.io https://protocols.io/view/bio-cn-redor-dd7f29jn
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 20, 2025
Protocol Integer ID: 100295
Keywords: CN-fsREDOR
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose
13C/15N frequency selective REDOR for distance measurement.
Scope
Intermolecular or intramolecular 13C-15N distance measurement up to ~6 Å range. The selectivity of recoupling relies on 13C and 15N chemical shift resolution.
Checklist for setting CN-fsREDOR experiment:
- Load parameters file or previous standard experiment acquired using the same spectrometer, probe, and spinning rate (if exist).
- Updated the optimized parameters (p14, plw14, p13, plw13, p15, plw15, p43, spnam41, const41, spnam31, cnst31, cpdprg4, p24, cnst24, spnam38, p38, cnst38, spnam58, p58, and cnst58).
- Set flags for soft pulses (L3 and L5)
- Set 13C and 15N frequencies for acquisition and selective pulses (o1p, o2p, o3p, cnst16, cnst17)
- Set acquisition time (aq) and spectral width (sw) for direct dimension.
- Set recycle delay (d1)
- Set array to acquire psudo-2D dataset (L0 and L8)
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 6.8 to find corresponding parameter names if using other pulse sequences.
Materials
Definitions:
| Term | Definition | |
| REDOR | Rotational-echo double-resonance | |
| fs | Frequency selective | |
| CP | Cross Polarization | |
| MAS | Magic Angle Spinning |
Instrument: The CN-fsREDOR 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 result, this experiment should be executed after properly adjusting the shimming and magic angle.
Sample:
- Type: Peptide, protein, or small molecules with resolved 13C and 15N resonances.
- Labeling: U-13C,15N labeling (small peptide or molecules), or sparsely labeled proteins.
- Minimum amount: ~ 50 nmol for 1.6 mm rotor
CN-fsREDOR 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: CN-fsREDOR):
Pulse parameters:
1H 90o: (p14, plw14, cnst14) = (2.5 µs, 33W. 100 kHz)
13C 90o:(p13, plw13, cnst13) = (2.5 µs, 140W, 100 kHz)
15N 90o: (p15, plw15, cnst15) = (5 µs, 125W, 50 kHz)
HC CP: [p43, cnst41(1H), cnst31(13C)]
=[1 ms, 88 kHz (tan70to90.1000), 60 kHz (cw)]
1H-decoupling - (CPDPRG4, p24, cnst24)
= (SPINAL64_p24, 5.2 ms, 90 kHz)
13C-soft pulse - [p38, spnam38, cnst38]
= [7.5 ms, Rsnob.1000, 0.28 kHz]
13C frequency: [cnst16, cnst17]
= [100 ppm, 175.20 ppm]
Acquisition parameters:
MAS rate (wr) in Hz: cnst20 = 13.333 kHz
Rotor period (wr): d20 = (1/cnst20) =75.0 ms
1H carrier frequency: O2p = 6 ppm
15N carrier frequency: O3p=118 ppm
Number of scans: ns = 8
Recycle delay: d1 = 1 sec
13C Direct dimension: t2 or F2:
Carrier frequency: O1p = 100 ppm
Acquisition time: aq(F2) = 30.9 ms
Spectral width: sw(F2) = 663.0 ppm
Experimental time = 15 min
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).
Temperature: As determined for optimal sample sensitivity and resolution.
Below optimizations are required before setting up the CN-fsREDOR experiment (parameters needs to be updated are shown in parenthesis). Example SOP for each optimization is in 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). Also note that the soft pulse duration (p38 or p58 must be rotor synchronized, i.e., 2mτr).
- Calibration of 1H, 13C, and 15N solid pulses (p14, plw14, p13, plw13, p15, and plw15)
- Optimization of HC CP (p43, spnam41, const41, spnam31, and cnst31)
- Optimization of high-power 1H decoupling (cpdprg4, p24, and cnst24)
- Optimization of 13C selective pulse (spnam38, p38, cnst38)
- Optimization of 15N selective pulse (spnam58, p58, cnst58)
Setup time: ~ 15 min, presuming all required optimizations/calibrations are done.
Procedure
Procedure
Load pulse program and parameter set.
If a previous CN-fsREDOR experiment acquired using the same setup is available (i.e., same spectrometer, probe, 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 “CN-fsREDOR” as PULPROG. Type “rpar”, then load file “CN-fsREDOR-NAN_par”.
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 flags properly to switch pulse sequences.
The pulse sequence has option to turn on/off the soft pulses for 13C/15N channels (i.e., L3 for 13C and L5 for 15N. 0 is off and 1 is on). If the soft pulses are turned off, then regular REDOR (with non-selective hard pulses) will be executed.
Set frequency parameters
For 1H, set the carrier frequency at ~5 ppm. On Topspin, type “O2p”, then input the value.
For 13C, there are 3 frequency related parameters. First is carrier frequency “O1p” which should be either the center of the 13C spectrum (i.e., 100 ppm for peptide), or the optimized one for 1H-13C CP transfer. Second is cnst16 which should be always the same as “O1p”. Third is cnst17 which should match the chemical shift of the 13C resonance being selected.
For 15N, set the carrier frequency “O3p” at the chemical shift of the 15N resonance being selected.
Set the acquisition parameters for direct dimension t1 (below parameters are typical for peptide samples).
1. Acquisition time: depending on 13C T2, default is ~15 ms
2. Spectral width: ~ 300 ppm.
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 use recycle delay less than 1s.
Acquire reference spectra and dephasing spectra, and store in one dataset.
The parameter to switch between reference and dephasing spectra is L0 (L0=0 is referencing, L0=1 is dephasing). L8 is the number of REDOR blocks. The REDOR mixing is calculated using L8*τr*2 (i.e., if spinning at 20kHz with L8 =16. Then the REDOR mixing time is 16*(1/20000)*2s = 1.6ms). See below schematic pulse sequence at section 6.10 for more details.
Acquire a spectrum with L8=0 and L0=0, then phase it properly.
Type “popt” to open an optimization window. In this window, select “store as 2D data (ser file)” and “Correlate 2D Container with experiment” options. L0 and L8 will be arrayed together so add one more row by press “add parameter” button. Then set the two arrays as shown below. The only values can be changed are the start and end value of L8, and the increment (highlighted by red dots). Press “Start optimize” to initiate the acquisition.
The above setting will acquire the reference and dephasing spectra for a L8 value, then increasing L8. The output result will look like below.
Topspin will output a pseudo 2D dataset named as “#999” (i.e., if the experiment number is 201, then the 2D dataset will be named as 201999). Open the dataset, then type “xf2” to process the direct dimension. Then locate the mouse curser to the position of the peak to find the slice number (in below example, the slice index is 6334). Or find the range of an integral (in below example, the range is ~6200 to ~6500).
If peak heights are needed, then type “rsc # #” (in above example, type “rcs 6334 6334”). This will generate a 1D dataset for the slice with this index number. It looks like below. The top points correspond to the reference spectra, and the bottom points correspond to the dephasing spectra.
If integrals are needed, type “f1sum”. It will open a window as below. Select “calculate sum” option, then specify the first row, last row, and destination number. In below example, it will calculate sum from slice 6200 to 6500, then store data into a dataset named as 6334. This will generate a similar 1D dataset as shown above.
Once get the 1D dataset, type “convbin2asc”. It will generate a txt file termed “ascii-spec.txt” in the raw data folder (i.e., in above example, the txt file will be in the /201999/pdata/6334 folder). Only the first two columns of this file are useful. The first column of the data file is the index of data point, and the second column of the data file is the peak intensity.
Protocol references
Reference: Jaroniec, et al. Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate 13C−15N distance measurements in uniformly 13C, 15N-labeled peptides. J. Am. Chem. Soc. 2001, 123, 15, 3507–3519 https://doi.org/10.1021/ja003266e