Feb 19, 2025
Bio - hNH 2D R1ρ
This protocol is a draft, published without a DOI.
- Songlin Wang1
- 1NMRFAM, University of Wisconsin-Madison
Protocol Citation: Songlin Wang 2025. Bio - hNH 2D R1ρ . protocols.io https://protocols.io/view/bio-hnh-2d-r1-dd7g29jw
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: 100296
Keywords: Pseudo-3D hNH experiment with 15N T1ρ measurement
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose
Pseudo-3D hNH dipolar correlation experiment with 15N T1ρ measurement at fast spinning rate for mapping the site-specific dynamics.
Scope
Site-specific dynamic measurement of protein using 1H detection.
Materials
Definitions:
| Term | Definition | |
| hNH | CP based HN 2D correlation experiment | |
| CP | Cross Polarization | |
| MAS | Magic Angle Spinning |
Instrument: The hNH2d-T1ρ Pseudo-3D dataset in this SOP was acquired using a 900MHz 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: 40 kHz
Sample: 25% 1H back exchanged, 15N, 13C, 2H -GB1
Pulse Parameters:
1H 90°: (p14, plw14, cnst14) = (2.5 μs, 100W, 100 kHz)
13C 90°: (p13, plw13, cnst13) = (5 μs, 108 W, 50 kHz)
15N 90°: (p15, plw15, cnst15) = (9.6 μs, 100W, 26.0 kHz)
HN CP: [p45, spnam41, cnst41, spnam51, cnst51]
= [2.50 ms, tan70to100, 36.5 kHz, cw, 10 kHz]
NH-CP: [p54, spnam42, cnst42, spnam52, cnst52]
= [1.25 ms, tan70to100, 31.5 kHz, cw, 10 kHz]
15N spin-lock: [p59, cnst59]
= [vplist, 2.5 kHz]
1H-decoupling - (CPDPRG4, p24, cnst24)
= (SPINAL64_p24, 46 μs, 10 kHz)
13C-decoupling - (CPDPRG3, p23, cnst23)
= (WALTZ16_p23, 100 μs, 2.5 kHz)
15N-decoupling - (CPDPRG5, p25, cnst25)
= (WALTZ16_p25, 100 μs, 2.5 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 = 40
Rotor period (tr): d20 = (1/cnst20) = 25 μs
Number of scans: ns = 8
Recycle delay: d1 = 1.5 sec
1H Direct dimension: t3 or F3:
Carrier frequency: O1p = 9 ppm
Acquisition time: aq(F3) = 41 ms
Spectral width: sw(F3) = 111.14 ppm
15N indirect dimension: t2 or F2:
FnMode: States-TPPI
Carrier frequency: O3p = 118 ppm
Maximum evolution time: aq(F2) = 50 ms
Spectral width: sw(F2) = 43.869 ppm
Dwell time: IN_F (F2) = 250 μs
Total t2 points: TD (F2) = 400
Spin-lock dimension: F1:
FnMode: QF
Total t1 points: TD (F1)=9
vplist: 0.1, 36000, 72000, 108000, 144000, 216000, 288000, 360000, 540000 μs
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. τ
Below optimizations are required before setting up the Pseudo-3D hNH dipolar correlation experiment with 15N T1ρ measurement (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_T1rho” pulse program and parameter set “T1p-hNH-NAN_par”.
Use Topspin command “edc” to open a new experiment.
Input “hNH2d_T1rho” as PULPROG
Type “rpar”, then load file “T1p-hNH-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.
Follow SOP to optimize and set all parameters for hNH2d experiment. Note that the F3 dimension in this pseudo-3D corresponds to F2 dimension of hNH 2D, and the F2 dimension in this pseudo-3D corresponds to F1 dimension of hNH 2D.
Set the spin-lock power.
The parameter for spin-lock rf amplitude is cnst59. The unit is kHz. The default power is 10 kHz.
The signal decay will be slower as the spin-lock power increase when the power is smaller than half of the spinning speed (1/2 ωR, Bloch-McConnell relaxation), while the signal decay will be dramatically faster when the spin-lock power is close to the spinning speed (ωR, near-rotary-resonance relaxation). Note that this is a general observation since the signal decay also depends on the dynamic of the sample.
When set cnst59, check plw59 as well which corresponds to the rf power in unit of Watts. Note that the spin-lock duration could be long (e.g. a few hundreds milliseconds), so using high rf power for spin-lock may damage the sample and probe. So, the plw59 should not be a large number (<10W is recommended for sample safety if long spin-lock durations are used). Please consult with your facility staff to find out the best combination of the spin-lock power and duration.
Make a vplist for the durations of spin-lock
Input a list name in “VPLIST”. For example, T1rho_SW is the vplist for the example dataset.
After input a name, press the “E” button to edit the list. Input all the spin-lock durations for the T1ρ measurement. Note that the unit is micro-second, and each number is one line. See below example. Save and quite after that.
Set F1 dimension regarding the vplist.
Set FnMODE as “QF”
Set TD as the number of spin-lock durations in the vplist (e.g. 9 for the above example).
The experiment will generate a series of hNH 2D spectra with different spin-lock durations and store them in a Pseudo-3D dataset. The number of hNH 2D spectra corresponds to the number of spin-lock durations in the vplist.
Determine experiment time
Minimal number of scans (phase cycle limited): 4
Note that a decrease of the hNH 2D sensitivity is expected as the spin-lock duration increase. For better sensitivity, adjust measurement time as required by incrementing number of scans in multiples of 4.
Validation
Start the experiment and monitor the first ~20-30 rows.
Process the first hNH 2D spectrum to check for adequate signal correctly arraying indirect dimension.
Protocol references