Dec 19, 2024

Public workspaceNOESYHSQC_15N_3D.nan

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l62xzzgqe/v1
NOESYHSQC_15N_3D.nan
  • NAN KB1,
  • Alex Eletsky2,
  • John Glushka2
  • 1Network for Advanced NMR ( NAN);
  • 2University of Georgia
NAN-KB UGA
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l62xzzgqe/v1
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2024. NOESYHSQC_15N_3D.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62xzzgqe/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: 100624
Keywords: protein nmr, assignment, backbone, amides, 15N, hsqc, chain resonance assignment of asn, linked 1h spin, backbone resonance assignment, 1h spin, hsqc pulse sequence with sensitivity enhancement, hsqc pulse sequence, backbone amide, systems 15n noesy, larger systems 15n noesy, noe correlation, sequential spin system, spin, chain resonance assignment, remote 1h spin, 3d noesy, 15n, he1 of trp ring, asn, pulse sequence, edited 3d noesy, resonance assignment, exchanging he spin, certain amino acid type, chemical shifts along the third dimension
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 an 15N-edited 3D NOESY-HSQC pulse sequence with sensitivity enhancement, gradient coherence selection and water flip-back, using either uniform or non-uniform sampling (NUS). This produces a 3D phase-sensitive dataset that provides NOE correlations between N-linked 1H spins and remote 1H spins resolved according to 15N chemical shifts along the third dimension (F2). The N-linked 1H spins typically include backbone amides, side-chain -CONH2 groups of Asn/Gln, and HE1 of Trp rings. In some cases, signals from slow exchanging HE spins of Arg and HD2 or HE1 of His can be observed as well.

This pulse sequence can be used for:
  • Aiding in backbone resonance assignment - sequential spin system linking can be validated by matching lineshapes of HA-HN, and HN-HN NOE cross-peaks.
  • Side-chain resonance assignment of Asn/Gln -CONH2 groups, Trp HE1/NE1, Arg HE/NE.
  • Generating distance restraints for structure calculation based on NOE cross-peak integrals or intensities

Required isotope labeling: U-15N, or U-15N,13C. Also suitable for samples with selective ("sparse") 15N-labeling of certain amino acid types.

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

Field strength preference: highest available field strength is preferred for better sensitivity and peak dispersion.

It uses a pulseprogram 'noesyhsqcfpf3gpsi3d.nan' modified from the original Topspin version.
Attachments
Icon for biotop.pdf
biotop.pdf
2.5MB
Icon for NUS_poisson_gap_files.zip
NUS_poisson_gap_file...
12KB
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.

NOE peak intensities depend on the mixing time. Initially the buildup rate is linear, reaching saturation as the mixing time increases. The buildup rate and saturation onset depend on protein MW. Short mixing times would yield more accurate distance restraints, at the cost of lower S/N and the loss of valuable long-range NOE peaks that are important for structure calculation. Very long NOE mixing times would result in peak intensities being no longer sensitive to internuclear distances. In practice, structure calculation algorithms work well with distance restraints generated even from somewhat saturated NOE data. The NOE mixing time is thus usually set to compromise values, for example, ~100 ms for small ubiquitin-sized proteins (~8 kDa), or ~60-70 ms for 20-25 kDa systems.

Due to large number of expected peaks and high dynamic range (strong diagonal peaks and weak cross-peaks) NUS sampling amount should be ~20-30% provided there is sufficient S/N.

2D F1(Hnoe)-F3(HN) plane can be acquired by setting 2 TD to 1, and ZGOPTNS to "-DZERODWN".

2D F2(15N)-F3(HN) plane can be acquired by setting 1 TD to 1, and ZGOPTNS to "-DZERODWH -DZERO".
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 3D setup and non-uniform sampling (NUS) options and use of the bioTop module are described in more detail in the protocol HNCO_3D.nan.

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 15N NOESY-HSQC experiment
Join an existing dataset and experiment (e.g. 1D proton, 2D 15N HSQC, etc) for this sample.
Click on Acquire -> 'Create Dataset' button to open dataset entry box,
or type edc command.
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 NOESYHSQC_15N_3D_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.
Return to the 'Acquire' menu and click 'Tune' ( or type atma on command line).
Load pulse calibrations: use getprosol (step 2.1) or bioTop (steps 2.2)
Note
Loading the NOESYHSQC_15N_3D_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 ( step 2.1), 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 in dB (PLdB1)]

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.

Skip to step 2.3.
Go to 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
Examine parameters by typing 'eda', or select the 'Acqpars' tab and get the 'eda' view by clicking on the 'A' icon. This view shows the three dimensions, F3(1H), F2(15N) and F1(1H) in columns.

Parameters to check:
  • FnTYPE - 'traditional planes' or 'non-uniform sampling' ( see step 2.5 below )
  • NS - minimum 2; optimally 4 to suppress axial peaks in both indirect dimensions. Increase for for higher signal to noise ( S/N increases as square root of NS )
  • DS - 32-128 'dummy' scans that are not recorded (multiple of 32); allows system to reach steady state equilibration. Important for HSQC-based experiments due to heating from 15N decoupling during acquisition
  • 3 SW - 1H spectral width (~12-15 ppm, defined in BioTop)
  • 2 SW - 15N amide spectral width (~25-40 ppm, defined in BioTop)
  • 1 SW - 1H spectral width, indirect (NOE) dimension (usually same as 3 SW, ~12-15 ppm, defined in BioTop)
  • O1 - 1H H2O offset in Hz (calibrated with BioTop or calibo1p1)
  • O2P - 13C aliphatic+aromatic offset (~69 ppm, same as CNST60)
  • 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 (2 AQ ~23 ms)
  • 1 TD - Number of 1H indirect (NOE) dimension time domain real points (1 AQ ≤16 ms, limited by JHH couplings)
  • DIGMOD - 'digital' ( pulse sequence does not use acqt0 correction )
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.

  • CNST4 - one-bond 1JNH coupling effective value (≥93 Hz); used to calculate INEPT transfer delays. For high MW proteins, increase the value of CNST4 for shorter transfer delays and higher S/N.
  • D1 - 1-2 sec
  • D8 - NOE mixing time (~70-120 ms)
  • D24 - refocused INEPT transfer delay (<0.0027 s). Typically set to match D26 or 1/(4*CNST4), optimizing for backbone N-H at the expense of CONH2
  • 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)
  • CNST60 - 13C aliphatic+aromatic offset (~69 ppm, same as O2P, for 13C decoupling in F1(1H) Hnoe dimension, with ZGOPTNS -DLABEL_CN)
  • CNST61 - 13C CA+CO offset (~110 ppm, for 13C decoupling during 15N evolution, with ZGOPTNS -DLABEL_CN)
  • P21 - 15N 90º high power pulse (calibrated with BioTop)


ZGOPTNS flags:
  • -DLABEL_CN - enable 13C decoupling during indirect 1H and 15N evolution for 13C,15N-labeled samples (default)
  • -DZERO - Apply 0º phase shift for initial 1H pulse (instead of 45º default shift). Used for acquiring 2D F3(HN)-F2(15N) HSQC planes (optional)
  • -DZERODWH - zero-dwell sampling in indirect F1(1H) NOE dimension. Used for acquiring 2D F3(HN)-F2(15N) HSQC planes (optional)
  • -DZERODWN - zero-dwell sampling in F2(15N) dimension. Used for acquiring 2D F3(HN)-F1(Hnoe) planes (optional)

Configure NUS (non-uniform sampling) - optional

After all other acquisition parameters (especially spectral widths and time-domain points) are set, change the FnTYPE parameter to 'non-uniform sampling' (type 'eda' and select 'Experimental' to get correct parameter window).

Then select NUS in the 'eda' parameter winow and set the desired NusAMOUNT [%] sampling density.

For adequate reconstruction, number of NUS points should be larger than the number of expected peaks in the N-Hnoe planes. Due to large number of expected peaks and high dynamic range (strong diagonal peaks and weak cross-peaks) NUS sampling amount should be at least ~20-30%.

After this you have the option of using either the built-in sampling schedule generator in Topspin or a third-party one.

To use the built-in sampling schedule generator in TopSpin set the NUSLIST parameter to 'automatic'. The sampling schedule will then be generated at acquisition start, and will be purely random apart from point density weighting according to NusJSP and NusT2 parameters.

A better way to generate the sampling schedule is with nusPGSv8 AU program. This AU program uses NusAMOUNT and TD values of the current experiment to generate a random schedule with 'Poisson gap' point spacing, and offers additional options for point density weighting and sampling order. ( see protocol 'Poisson Gap NUS Acquisition Setup', and attached files 'nusPGSv8' and 'poissonv3'). To use this method, type 'nusPGSv8' on the command line. You can typically accept the default values in pop-up dialog windows, since they are suitable for most applications. A schedule will be generated and will be stored to the parameter NUSLIST.

If nusPGSv8 is not installed, copy the attached file 'nusPGSv8' to your user AU directory, /opt/topspin.X.X.X/exp/stan/nmr/au/src/user, and copy the binary file 'poissonv3' to /opt/topspinX.X.X/prog/bin.

Acquire and Process Data
Type 'expt' to calculate the expected run time.
Go to 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.

3D NUS data usually cannot be processed in Topspin without a separate license (see note in step 2.5). 2D planes with NUS or uniformly sampled can be processed in Topspin, though.

Also, a 2D NUS plane can be extracted from a completed 3D NUS data set with the command 'rser2d', and then processed within Topspin.

Full 3D NUS processing is usually performed using third-party processing software, such as NMRPipe together with specialized NUS reconstruction programs (SMILE, hmsIST, NESTA, etc.).
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

G.M. Clore (1989) Biochemistry 28 6150-6156. (Link to Article) D. Marion, L.E. Kay, S.W. Sparks, D.A. Torchia and A. Bax (1989) J. Am. Chem. Soc. 111 1515-1517. (Link to Article) E.R.P. Zuiderweg and S.W. Fesik (1989) Biochemistry 28 2387-2391. (Link to Article)

'nusPGSv8' written by Scott Anthony Robson 2013