Dec 19, 2024
HSQC_IPAP_15N.nan
- NAN KB1,
- Alex Eletsky2,
- John Glushka2
- 1Network for Advanced NMR ( NAN);
- 2University of Georgia
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2024. HSQC_IPAP_15N.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn63rql5d/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 25, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 100627
Keywords: protein nmr, assignment, backbone, amides, 15N, hsqc, hsqc pulse sequence, hsqc pulse sequence for for measurement, proteins with additional 2h labeling, hsqceft3gpiaphsisp, 2d 15n ipap, size protein, protein, pulse sequence, dipolar coupling, 15n, topspin library pulseprogram, 2h labeling, additional 2h labeling, pulseprogram
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 a 2D 15N IPAP-HSQC pulse sequence for for measurement of 1JH,N scalar and 1DH,N dipolar couplings along F1(15N), suitable for small to medium-size proteins (< 25 kDa. It yields two 2D sub-spectra.
15N labeling is required; can also be applied to proteins with additional 2H labeling.
It uses the Topspin library pulseprogram 'hsqceft3gpiaphsisp'.
Guidelines
Number of directly acquired points (2 TD) should be set to keep acquisition time t2,max (2 AQ) ~50-120 ms. For large proteins (~25 kDa) set closer to the lower range, higher values for small proteins.
Due to the inner loop cycling, appropriate setting of 1 TD (15N) points depends on the TopSpin version. TopSpin 3.x does not explicitly compensate for the inner loop, thus 1 TD should be set to twice the expected amount (twice the expected time resolution '1 AQ'). In TopSpin 4.x the inner loop does not count towards 1 TD, and '1 AQ' represents the true time resolution.
NUS sampling is usually not required for 2D experiments, since time savings are small, unless running multiple 2D experiments. If using NUS keep sampling amount ~30-50%.
For samples with 13C labeling use -DLABEL_CN flag to enable 13C decoupling during 15N evolution. 13C channel offset O2P should be set ~110 ppm (middle of 13C aliphatic and 13CO shift range).
For processing in TopSpin use 'split 2' command to extract individual sub-spectra.
Troubleshooting
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
- Acquisition Setup Workflow, Solution NMR Structural Biology
- PRESAT_bio.nan
- HSQC_15N.nan
- HNCO_3D.nan
- Biotop-Calibration and Acquisition setup
Create HSQC_IPAP_15N experiment file
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 HSQC_IPAP_15N_xxx.par, where xxx=900,800 or 600.
Click OK at bottom of window to create the new EXPNO directory.
If not done, tune Nitrogen and Carbon channels:
-on the Acquire menu, click Tune ( or type atma on command line).
Calibrate pulses and adjust parameters
Note
Loading the HSQC_IPAP_15N_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.
Using 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 'edprosol' table, and are assumed to be sufficiently accurate.
Skip to step 2.3.
Use 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 parameters' and attached Bruker manual 'biotop.pdf' for details.
Inspect 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 two dimensions, F2 (1H) and F1 (15N) in columns.
Parameters to check:
2 SW: 1H spectral width (~12-15 ppm, defined in BioTop)
1 SW: 15N amide spectral width (~25-40 ppm, defined in BioTop)
O1P: 1H offset defaults to 4.7ppm ( calibrated with bioTop)
O2P: 13C offset (~110 ppm, for decoupling when ZGOPTNS -DLABEL_CN)
O3P: 15N amide offset (~115-120 ppm, defined in bioTop)
2 TD: Number of 1H time domain real points (~1024-2048, preferably 2N, keep 3 AQ at ~50-120 ms)
1TD: Number of 15N time domain real points, 1 AQ at ~30-60 ms (TopSpin 4.x) or ~60-120 ms (TopSpin 3.x). In TopSpin 3.x inner IPAP loop counts toward the TD points.
DIGMOD - 'baseopt' (zero 1st order phase correction)
Then examine the parameters in Acqpars 'ased' mode (click 'pulse' icon), or type 'ased';
Most of the default parameters are suitable, however it's useful to compare values in the fields against those proposed by the original parameter file and the pulseprogram.
CNST4 - 1JNH value (≥93 Hz, used to calculate INEPT transfer delays, can be optimized using popt in 1D mode to maximize S/N)
D1: recyle delay (~1 s for protonated samples, ~2-3 s for perdeuterated samples)
D21: IPAP filter delay (5.3 ms)
DS: 32-128 'dummy' scans that are not recorded ; allows system to reach steady state equilibration.
NS: multiple of 4; increase for increased signal to noise ( S/N increases as √NS )
ZGOPTNS flags
-DLABEL_CN : enable 13C decoupling during 15N evolution for 13C,15N-labeled samples (default)
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)
P3 - 13C 90º high power pulse (calibrated with BioTop)
P21 - 15N 90º high power pulse (calibrated with BioTop)
Acquire and Process Data
Type 'rga' or click on 'Gain' in Topspin Acquire menu to execute automatic gain adjustment.
Type 'expt' to calculate the expected run time
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 planes with NUS or uniformly sampled can be processed with Topspin: xfb
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
R.T. Clubb, V. Thanabal & G. Wagner, J. Magn. Reson. 97, 213-217 (1992)
L.E. Kay, G.Y. Xu & T. Yamazaki, J. Magn. Reson. A109, 129-133 (1994)
'nusPGSv8' written by Scott Anthony Robson 2013