Dec 19, 2024

Public workspaceHNCA_3D.nan

DOI
dx.doi.org/10.17504/protocols.io.8epv5r1m4g1b/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.8epv5r1m4g1b/v1
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2024. HNCA_3D.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r1m4g1b/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: April 16, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 98277
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 a 3D HNCA 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 correlates backbone 1HN and 15N resonances of residue (i) with 13CA of residues (i) and (i-1). The sequential (i-1) 13CA peaks are typically weaker than intra-residue (i) 13CA peaks.

3D HNCA it often optional for backbone resonance assignment. It may be useful for challenging cases, by complementing HNCACB if it has low S/N. HNCA has better sensitivity than HNCACB and HN(CA)CO, though lower than HNCO. However, 13CA shifts alone are rather limited in the ability to differentiate residue types - usually only Gly residues can be distinguished from non-Gly.

Required isotope labeling: U-15N,13C. Samples with additional 2H labeling should use HNCA or TROSY-HNCA versions with 2H decoupling.

Optimal MW is ≤ 25 kDa. Larger systems generally benefit from 3D TROSY-HNCA versions. Compare 2D H-N planes of HNCO and TROSY-HNCO to check which one yields better S/N.

This pulse sequence can be used for:
  • spin system identification
  • resolving signal degeneracy in 2D 15N HSQC.
  • backbone resonance assignment: sequential linking of spins systems based on 13CA peaks matching (in conjunction with 3D CBCA(CO)NH, HN(CO)CA or HN(CO)CACB). Complements sequential linking of spins systems via HN(CA)CO and HNCACB.
  • resonance assignment of 13CA spins
  • chemical shift-based secondary structure prediction (e.g. TALOS), together with other resonances

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

It uses the standard Bruker Topspin pulseprogram 'hncagp3d'
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.

Since the F2(15N) is a constant-time dimension, number of acquired points 2 TD should be should be set to the largest possible value for optimal S/N. The maximum 2 TD value is 2*int(d30/in30), where d30 is the decremented delay, and in30 is the decrement value. Both d30 and in30 are computed internally according to pulse program code and their values are shown in ased view. Note that in30 = in10 = 1(4*SW(N)). In TopSpin 4.x the ased command would produce a warning if 2 TD is set too large and reset the internal td2 parameter to the maximum possible value.

As 3D HNCA only has 2 peaks per residue, NUS sampling amount can be as low as 5-10%, provided the sample delivers sufficient S/N.

Individual F1(CO)-F3(HN) or F2(N)-F3(HN) 2D planes can be acquired by setting, respectively, 2 TD or 1 TD to 1.

To check whether TROSY-HNCA or regular HNCA yields better S/N for your sample at a particular field strength, record and inspect the respective 2D F2(N)-F3(HN) planes of TROSY-HNCO or regular HNCO using identical spectral parameters.
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 and 2D 13C HSQC are also recommended, according to protocols HSQC_15N.nan and HSQC_CT_13C_ALI.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. HSQC_CT_13C_ALI.nan
  5. HNCO_3D.nan
  6. Biotop-Calibration and Acquisition setup

Create HNCA experiment file
Create HNCA experiment file
Join an existing dataset and experiment (e.g. 1D proton, 2D 15N HSQC, 2D 13C 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 HNCA_3D_xxx.par, where xxx=900,800 or 600*.


*parameter file name may be different
Click OK at bottom of window to create the new EXPNO directory.
It will be the active experiment in the acquisition window and should now be listed on your data browser.
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)
Load pulse calibrations: use getprosol (step 2.1) or bioTop (steps 2.2)

Note
Loading the HNCA_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.

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
Selecting the 'Acqpars' tab will show lists of common and pulse program specific acquisition parameters.
First view the 'eda' list by clicking on the 'A' icon or typing eda. This view shows the three dimensions, F3 (1H) and F2 (15N) and F1 (13C) in columns.


Parameters to check:
  • FnTYPE - 'traditional planes' or 'non-uniform sampling' ( see step 2.5 below )
  • NS - minimum 2; 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 16); allows system to reach steady state equilibration. Important for HSQC-based experiments due to heating from 15N decoupling during acquisition
  • SW [ppm] - 1H(F3) ~12-15 ppm; 15N (F2) ~25-40 ppm; 13CA (F1) ~32 ppm; defined in bioTop
  • O1 - 1H H2O offset in Hz (calibrated with BioTop or calibo1p1)
  • O2P - 13CA offset (~54 ppm, middle of 13CA range, defined in bioTop)
  • O3P - 15N amide offset (~115-120 ppm, defined in bioTop)
  • 3 TD - Number of 1H time domain real points (~1024-4096, preferably 2N, keep 3 AQ at ~50-120 ms)
  • 2 TD - Number of 15N time domain real points (2*int(d30/in30); 2 AQ ~24ms, CT dimension)
  • 1 TD - Number of 13CA time domain real points ( 1 AQ at ~12 ms, limited by 1JCA,CB coupling)
  • 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 values should be appropriate, however it's useful to compare them against suggestions in the pulseprogram comments.

  • D1 - 1-2 sec
  • 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)
  • CNST21 - 13CO shift for decoupling ( ~173 ppm, defined in bioTop)
  • P21 - 15N 90º high power pulse (calibrated with BioTop)
Configure NUS (non-uniform sampling) - 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).

Navigate down to the 'NUS' section in the 'eda' parameter window and set the desired NusAMOUNT [%] sampling density.

For adequate reconstruction, number of NUS points should be larger than the number of expected peaks. 3D HNCA yields 2 peaks per residue, thus a 100-residue protein would require more than 200 NUS points.

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
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.
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

L.E. Kay, G.Y. Xu & T. Yamazaki, J. Magn. Reson. A109, 129-133 (1994)

J. Schleucher, M. Sattler & C. Griesinger, Angew. Chem. Int. Ed. 32, 1489-1491 (1993)

S. Grzesiek & A. Bax, JMR 96, 432-440 (1992)

'nusPGSv8' written by Scott Anthony Robson 2013