Jan 14, 2025

Public workspaceT1_15N_HSQC.nan

DOI
dx.doi.org/10.17504/protocols.io.ewov19qw7lr2/v1
  • NAN KB1,
  • Alex Eletsky2,
  • John Glushka2
  • 1Network for Advanced NMR ( NAN);
  • 2University of Georgia
NAN-KB UGA
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DOI: dx.doi.org/10.17504/protocols.io.ewov19qw7lr2/v1
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2025. T1_15N_HSQC.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19qw7lr2/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: Working
We use this protocol and it's working
Created: May 28, 2024
Last Modified: January 14, 2025
Protocol Integer ID: 100693
Keywords: protein nmr, assignment, backbone, amides, 15N, hsqc
Funders Acknowledgements:
NSF
Grant ID: 1946970
Disclaimer
This protocol is part of the knowledge base content of NAN: The Network for Advanced NMR ( https://usnan.nmrhub.org/ )
Specific filenames, paths, and parameter values apply to spectrometers in the NMR facility of the Complex Carbohydrate Research Center (CCRC) at the University of Georgia.
Abstract
This protocol describes running 15N T1 HSQC experiments with temperature compensation, sensitivity enhancement, gradient coherence selection, water flip-back, and optional 13C decoupling. This is a pseudo-3D experiment, with the third dimension F1(T1) sampling a list of T1 relaxation delays provided in a separate text file. 1H BIP shaped 180º pulses are used to suppress cross-relaxation and cross-correlated relaxation between 1H and 15N spins during T1 relaxation delay.

Temperature compensation involves application of pulses prior to recycle delay delivering the same total RF power for each T1 delay. Total acquisition time thus strongly depends on the maximum T1 delay.

This experiment is primarily used for studying protein backbone dynamics on the picosecond to nanosecond scale in combination with 15N T2 HSQC, {1H}-15N HNOE, etc.

Required isotope labeling: U-15N, or U-15N,13C (with or without 2H). Also suitable for samples with selective ("sparse") 15N-labeling of certain amino acid types.

Optimal MW is ≤ 30-35 kDa. For larger systems 15N T1 TROSY may be more appropriate.

Field strength preference: detailed analysis of protein backbone dynamics often requires measurement of 15N relaxation rates at multiple fields.

It uses the Topspin library pulseprogram 'hsqct1etf3gpsitc3d'.


Attachments
Icon for biotop.pdf
biotop.pdf
2.5MB
Guidelines
The number of directly acquired points (3 TD) should be set so the acquisition time t3,max (3 AQ) is between ~50 ms (for larger proteins ~25 kDa) and ~120 ms (for smaller proteins). Longer times may cause excessive probe and sample heating during 15N decoupling, and resolve undesirable 3JHN,HA splittings.

"Effective" 1JNH coupling value CNST4 determines the length of the INEPT transfer delays. For larger proteins CNST4 can be increased (i.e. > 92 Hz) to reduce losses due to relaxation. CNST4 can be optimized by arraying using popt with 15N HSQC experiment in 1D mode.

For samples with 13C labeling use -DLABEL_CN ZGOPTNS flag to enable 13C decoupling during 15N evolution. CNST21 and CNST22 are used to define the 13C channel offset for decoupling.

Since 15N T1 HSQC is a quantitative experiment, it is recommended to use longer D1 recycle delays and a sufficient number of dummy scans DS. 1H spectral width 3 SW should be set a few ppm wider than required by the peak dispersion to allow for "empty" noise regions for proper baseline correction during FT processing.

T1 relaxation delays should be multiples of 10 ms, and should include ~8 points or more. The shortest delay should be ≥20 ms, to avoid loop counter being less than 1. For optimal fitting precision, the longest delay should be at least as long at the expected T1 relaxation time (usually in the range of 1-3 s). This may vary from protein to protein, as T1 increases with MW. Due to temperature compensation, the longest delay also determines the overall experiment time, thus it should not be set excessively long.


Before start
A sample must be inserted in the magnet either locally by the user after training, or by facility staff if running remotely.

This protocol requires a sample is locked, tuned/matched on 1H, 13C and 15N channels, and shimmed. At a minimum, 1H 90° pulse width and offset O1 should be calibrated and a 1D proton spectrum with water suppression has been collected according to the protocol PRESAT_bio.nan. Prior acquisition of a 2D 15N HSQC is also recommended, according to protocol HSQC_15N.nan.

General aspects of the use of the bioTop module are described in more detail in the protocols listed below.

It is recommended to calibrate 1H carrier offset, 1H H2O selective flip-back pulse, as well as 1H, 13C, and 15N 90° pulse widths using the "Optimization" tab of BioTop. Alternatively, 1H 90° pulse width and offset can be calibrated using other methods, such as pulsecal or calibo1p1. Additional parameters, like 15N and 13C offsets and spectral width can be either optimized or manually entered in the "Optimization" tab of BioTop. Note that since BioTop optimizations are saved in the dataset folder, all experiments should be created under the same dataset name when using BioTop for acquisition setup.

Refer to protocols
  1. Acquisition Setup Workflow, Solution NMR Structural Biology
  2. PRESAT_bio.nan
  3. HSQC_15N.nan
  4. HNCO_3D.nan
  5. Biotop-Calibration and Acquisition setup

Create T1_15N_HSQC experiment
Create T1_15N_HSQC experiment
Start with existing dataset containing 1D proton or 15N HSQC data for this sample.
Open create dataset window with edc command or through menu.
Edit text in Title box.
To select starting parameter set, check 'Read parameterset' box, and click Select.
For standard NAN parameter sets, change the Source directory at upper right corner of the window:
Source = /opt/NAN_SB/par
Click 'Select' to bring up list of parameter sets.
Select T1_15N_HSQC_xxx.par, where xxx=900,800 or 600.
Click OK at bottom of window to create the new EXPNO directory.
If not previously done, tune Nitrogen and Carbon channels. Return to the 'Acquire' menu and click 'Tune' (or type atma or atmm on command line). Re-shim after tuning.
Load pulse calibrations: use getprosol (step 2.1) or bioTop (steps 2.2)
Load pulse calibrations: use getprosol (step 2.1) or bioTop (steps 2.2)
Note
Loading the T1_15N_HSQC_xxx.par parameter set enters the default parameters into the experiment directory. While they represent a good starting point, they may not be fully optimal or accurate for your particular sample or spectrometer hardware. The probe- and solvent-specific parameters, specifically the 1H 90° pulse length, and possibly the 13C and 15N 90° pulse lengths, along with other dependent pulse widths and powers may need to be updated.

There are two ways of automatically updating experimental parameters:
1) Use getprosol command, which typically only updates proton pulse widths and power levels. It is most useful for running routine experiments using the default parameters.
2) Or use bioTop module that organizes calibrated and defined parameters for a dataset.

Loading pulse widths and power levels with getprosol:

Use the calibrated proton P1 value obtained from the proton experiment ( protocol PRESAT_bio.nan) and note the standard power level attenuation in dB for P1 (PLW1); otherwise type calibo1p1 and wait till finished.

Then execute the getprosol command:
getprosol 1H [ calibrated P1 value] [power level for P1]
e.g. getprosol 1H 9.9 -13.14.
Where for example, the calibrated P1=9.9 at power level -13.14 dB attenuation
This also loads 15N and 13C pulse widths and power levels from the PROSOL table, and are assumed to be sufficiently accurate.

Go togo to step #2.3 If not using BioTop
Loading experimental parameters from BioTop:

If you previously performed parameter calibrations using the "Optimizations" tab of the BioTop GUI, or entered parameters manually in the "Optimizations" tab, you can type btprep at the command line.

See protocol 'BioTop: calibration and acquisition setup' and attached Bruker manual 'biotop.pdf' for details.
Inspect and adjust parameters
Inspect and adjust parameters
Examine parameters by typing 'eda', or select the 'Acqpars' tab and get the 'eda' window by clicking on the 'A' icon. This view shows the three dimensions, F3 (1H), F2(15N), and F1(T1) in columns.

Parameters to check:
  • DS - 32-128 'dummy' scans that are not recorded; allows system to reach steady state equilibration.
  • NS - multiple of 8; increase for increased signal to noise ( S/N increases as √NS )
  • 3 SW - 1H spectral width (~12-15 ppm, defined in BioTop)
  • 2 SW - 15N amide spectral width (~25-40 ppm, defined in BioTop)
  • O1 - 1H H2O offset in Hz (calibrated with BioTop or calibo1p1)
  • O2P - Not used. 13C offset set internally to the average of CNST21 and CNST22 (for 13C decoupling with ZGOPTNS -DLABEL_CN)
  • O3P - 15N amide offset (~115-120 ppm, defined in bioTop)
  • 3 TD - Number of 1H time domain real points (~1024-2048, preferably 2N, keep 3 AQ at ~50-120 ms)
  • 2 TD - Number of 15N time domain real points (keep 1 AQ at ~30-60 ms)
  • 1 TD - Number of T1 delay points (should match number of lines in VDLIST)
  • DIGMOD - 'baseopt' (zero 1st order phase correction in 1H)
Then examine the parameters in Acqpars 'ased' mode (click 'pulse' icon), or type ased.
Most of the default parameters should be appropriate, however it's useful to compare values in the fields against those proposed by the original parameter file and the pulseprogram comments.
General:
  • CNST4 - one-bond 1JNH coupling effective value (≥93 Hz); used to calculate INEPT transfer delays.
  • CNST11 - multiplicity factor; 4 - optimized for NH (default), or 8 - optimized for NH/NH2
  • CNST12 - multiplicity factor; 4 - optimized for NH (default), or 8 - optimized for NH/NH2
  • D1 - recycle delay (~2 s for protonated samples, ~3 s for perdeuterated samples). Usually set high for quantitative/relaxation experiments
  • VDLIST - T1 delay list file. Click on dots button '...' to select, or on 'E' to edit. Each delay must be a multiple of 10 ms; use at least 8 points. The longest delay should be at least as long as the expected T1 relaxation time (~1-3 s, may need to be adjusted depending on protein MW). Also determines the duration of temperature compensation.

ZGOPTNS flags
  • -DLABEL_CN - enable 13C decoupling during 15N evolution for 13C,15N-labeled samples (default)

Channel f1:
  • P1 - 1H 90º high power pulse (calibrated with calibo1p1 or BioTop)
  • SPdB1 - power level [dB] for 1H H2O flip-back shaped pulse (calibrated with BioTop)

Channel f2 (only for ZGOPTNS -DLABEL_CN):
  • CNST21 - 13CO offset (~173 ppm, calibrated with BioTop) for 13C decoupling
  • CNST22 - 13CA offset (~53 ppm, calibrated with BioTop) for 13C decoupling

Channel f3:
  • P21 - 15N 90º high power pulse (calibrated with BioTop)
Acquire and Process Data
Acquire and Process Data
Type 'expt' to calculate the expected run time
Go togo to step #2.3 If necessary to re-adjust parameters

Type 'rga' or click on 'Gain' in Topspin Acquire menu to execute automatic gain adjustment.
Type 'zg' or click on 'Run' in Topspin Acquire menu to begin acquisition.
You can always check the first FID by typing efp to execute an exponential multiplied Fourier transform. It will ask for a FID #, choose the default #1. You can evaluate the 1D spectrum for amide proton signal to noise and water suppression.
2D F2(15N)-F3(1H) and F1(T1)-F3(1H) planes can be processed with Topspin commands xfb and xf2, respectively.
Protocol references
J.Cavanaugh, W.Fairbrother, A.Palmer, N.Skelton: Protein NMR Spectroscopy: Principles and Practice.
Academic Press 2006 ; Hardback ISBN: 97801216449189, eBook ISBN: 9780080471037