Feb 19, 2025
Bio - hCC 2D DARR
This protocol is a draft, published without a DOI.
- Songlin Wang1
- 1NMRFAM, University of Wisconsin-Madison
Protocol Citation: Songlin Wang 2025. Bio - hCC 2D DARR. protocols.io https://protocols.io/view/bio-hcc-2d-darr-dd6r29d6
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: 100273
Keywords: hCC, DARR, CC2d
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose
hCC 2D homonuclear dipolar correlation experiment with DARR mixing at moderate spinning rate.
Scope
Residue type identification with short mixing times (10-50 ms); inter-residue correlations with moderate mixing times (50-200 ms); distance measurements with long mixing times (>200 ms).
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 "Materials" to find corresponding parameter names if using other pulse sequences.
Materials
Definitions:
| Term | Definition | |
| hCC | Homonuclear 2D correlation experiment with CP | |
| DARR | Dipolar assisted rotational resonance | |
| CP | Cross Polarization | |
| MAS | Magic Angle Spinning | |
| SSB | Spinning side-band |
Instrument: The CC 2D DARR 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 1.6 mm HCN probe. This experiment critically requires proper adjustment of the shimming and magic angle.
Sample:
- Type: Protein
- Labeling: U-13C,15N labeling (default), or 13C-glycerol skip-labeled.
- Minimum amount: ~50 nmol for 1.6 mm rotor.
hCC DARR 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: hCC-NAN):
Pulse Parameters:
1H 90°: (p14, plw14, cnst14) = (2.5 μs, 43W, 100 kHz)
13C 90°: (p13, plw13, cnst13) = (2.5 μs, 150 W, 100 kHz)
15N 90°: (p15, plw15, cnst15) = (5 μs, 115W, 50 kHz)
HN CP: [p43, cnst41 (1H), cnst31(13C)]
= [1 ms, 88 kHz (tan70to90.1000), 60 kHz (cw)]
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 =13.333 kHz
Rotor period (tr): d20 = (1/cnst20) =75.0 ms
1H carrier frequency: O2p = 6 ppm
Number of scans: ns = 4
Recycle delay: d1 = 1 sec
13C Direct dimension: t2 or F2:
Carrier frequency: O1p = 100 ppm
Acquisition time: aq(F2) = 15.4 ms
Spectral width: sw(F2) = 331.5 ppm
13C indirect dimension: t1 or F1:
Carrier frequency: O2p = 100 ppm
Maximum evolution time: aq(F1) = 12.8 ms
Spectral width: sw(F1) = 265.2 ppm
Total t1 points: TD (F1) = 1024
Complex t1 points: 512 (States-TPPI)
Experimental time = 2.5 hours
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 (~88.9 ppm in 13C to avoid SSB overlap):
13.333 kHz at 600 MHz
16.667 kHz at 750 MHz
20 kHz at 900 MHz
25kHz at 1.1 GHz
Note that the transfer efficiency of DARR decreases significantly when MAS > 30 kHz, and alternatives such as CORD or RFDR mixing are preferred at higher MAS rates
Temperature: As determined for optimal sample sensitivity and resolution.
The following optimizations are required before setting up the CC 2D DARR experiment (parameters requiring updates are shown in parentheses).
Example SOPs for each optimization are attached at "References". Note that RF amplitudes in this pulse sequence are defined in units of kHz rather than Watts (i.e., input kHz number for RF powers and the pulse sequence code will calculate the corresponding values in Watts for Topspin to use). Only the hard pulses for calibration need to be input in units of Watts (plw13, plw14, and plw15).
- Calibration of 1H, 13C, and 15N solid pulses (p14, plw14, p13, plw13, p15, and plw15)
- Optimization of HC CP (p43, spnam43, const41, and cnst31)
- Optimization of high-power 1H decoupling (cpdprg4, p24, and cnst24)
Setup time: ~ 10 min, presuming all required optimizations/calibrations are done.
Procedure
Procedure
Load pulse program and parameter set.
If a previous CC 2D DARR 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 “hCC-NAN” as PULPROG. Type “rpar”, then load file “hCC-NAN_par”.
Set the optimized pulse parameter values. The optimizations that need to be done and parameters need to be updated are listed at the "Before start" section under "Guidelines & Warnings".
Set the 1H carrier frequency at ~5 ppm, and 13C carrier frequency at ~100 ppm.
Ex: On Topspin, 1H carrier frequency “o2p”, and 13C carrier frequency “o1p”.
Set the spinning speed and DARR mixing.
Set the DARR 1H condition. The 1H rf power for DARR mixing should match the MAS rate. For the hCC NAN pulse sequence (hCC-NAN), below three parameters need to be changed. The spinning speed (cnst20, unit is Hz), 1H rf power for DARR mixing (cnst5, unit is kHz, should match the spinning speed), and the DARR mixing time (d5, see the next section for details).
Choose DARR mixing time
- For short range intra-residue correlations set the DARR mixing to 10-50 ms.
- For medium range inter-residue correlations set the DARR mixing to 50-200 ms.
- For long range (>5 Å) correlations set the DARR mixing to >200 ms.
- Optimal mixing times increase with magnetic field. Refer to standard data sets for guidance.
Set the acquisition parameters.
Direct dimension (t2)
- Acquisition time (AQ [sec]): depending on 13C T2, default is ~15 ms (alternative for 13C-glycerol labeling or crystalline samples: ~25 ms).
- Spectral width (SW [ppm]): ~ 300 ppm.
- Carrier frequency (O1P [ppm]): 100 ppm.
Indirect dimension (t1)
- Spectral width (SW [ppm]): coupled with dwell time, > 200 ppm is required to cover the full spectrum.
- Hypercomplex scheme (FnMODE): States-TPPI
- Increment of delay (IN_F [µsec]): 0.2 τR to 0.33 * τR, depending on magnetic field. (e.g., 25 µs at 600 MHz or 12.5 µs at 900 MHz). The spectral width must be an integer multiple of the MAS rate.
- Maximum evolution time (AQ [sec]): ~10 ms. (This presumes a CA T2 specification of 10 ms, recommend measuring CA T2 prior to this experiment).
- Number of rows (TD): coupled with increment of delay and maximum evolution time.
Set the recycle delay
Use recycle delay (d1) 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. Beware of heating limits for the probe and sample!
If duty cycle exceeds probe specification, an error should appear when attempting to start acquisition; if this is observed, increase the recycle delay until the error is resolved.
Determine experiment time
Minimal number of scans (phase cycle limited): 4.
Minimal measurement time: ~1 hr (depending on sample sensitivity).
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
Reference: Takegoshi, et al. 13C-1H dipolar assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 2001, 344, 631. https://doi.org/10.1016/S0009-2614(01)00791-6