Dec 20, 2024
hCCH-COSY_aro_3D.nan
- NAN KB1,
- Alex Eletsky2,
- John Glushka2
- 1Network for Advanced NMR ( NAN);
- 2University of Georgia
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2024. hCCH-COSY_aro_3D.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywj4qvx9/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 26, 2024
Last Modified: December 20, 2024
Protocol Integer ID: 100623
Keywords: protein nmr, assignment, backbone, amides, 15N, hsqc, aromatic 3d, aromatic 13c, aromatic resonance, 13c spin, dispersion of aromatic 13c, cosy pulse sequence, resonances within aromatic ring, assignment of aromatic resonance, cosy scheme, cosy dimension, aromatic ring, aromatic 1h polarization, aromatic spectral region, hsqc pair, resonance assignment of aromatic side, resonances in phe, hsqc peak, 3d phase, cb shifts from the backbone resonance assignment stage, cosy experiment, aromatic side, backbone resonance assignment, backbone resonance assignment stage, indirect 13c dimension, cosy, multiple 13c line, resonance, 1jcc coupling
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 (H)CCH-COSY pulse sequence optimized for the aromatic spectral region. It uses semi-constant time (SCT) evolution in both indirect 13C dimensions, and can be collected with a standard or a non-uniform (NUS) sampling scheme. This produces a 3D phase-sensitive dataset that correlates 1H and 13C resonances within aromatic rings of Trp, Phe and Tyr via 1JHC and 1JCC couplings.
Dimensions F3(1H) and F2(13C) represent an HSQC pair, while F1(13C) is the COSY dimension that exhibits multiple 13C lines for a given 13C HSQC peak. 3D (H)CCH-COSY scheme is usually preferred over a related 3D H(C)CH-COSY experiment, due to better signal dispersion of aromatic 13C as compared to 1H.
Aromatic 3D (H)CCH-COSY is used for resonance assignment of aromatic side-chain 1H and 13C spins, combined with 2D 13Caro CT-HSQC, 3D 13Cali-edited NOESY, and 3D 13Caro-edited NOESY. Assignment of aromatic resonances is usually performed after backbone and aliphatic side-chains assignment is complete.
The CA and CB shifts from the backbone resonance assignment stage along with HA and HB shifts from 3D HBHA(CO)NH are used to bootstrap the side-chain assignment process.
Required isotope labeling: U-15N,13C or U-13C. Not suitable for samples with additional 2H labeling due to starting with aromatic 1H polarization.
Optimal MW is ≤ 25 kDa.
Field strength preference: Highest available B0 field is preferred. Low dispersion of 13C resonances in Phe and sometimes Trp residues may lead to strong coupling artifacts at low fields.
It uses a pulseprogram 'hcchcoctgp3d2_aro.nan' modified from the original Topspin version.
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 during 13C decoupling, and resolve undesirable 3JH,H splittings.
If acquisition time in the indirect 13C dimensions is kept under 4.5 ms, it defaults to purely constant-time (CT) evolution. Higher evolution time settings automatically enable semi-constant time evolution. Maximum useful evolution time is limited to ~6 ms due to 1JCC couplings. It is recommended to utilize the full 6 ms resolution due to high sensitivity of aromatic (H)CCH-COSY.
Individual F2(13C)-F3(1H) and F1(13C)-F3(1H) 2D planes can be acquired by setting, respectively, 1 TD or 2 TD to 1.
Aromatic resonance assignment is usually initiated by identifying HD (Phe/Tyr) and HD1 (Trp) resonances among NOE cross-peaks within HB2/HB3 strips in 13Cali-edited NOESY and confirmed via "mirror" cross-peaks in 13Caro-edited NOESY. Alternatively, Yamazaki experiments, such as (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HDHE, can be used to identify aromatic 1H independently of the NOE data, though these experiments have very low sensitivity and are rarely required. Next, the corresponding CD (Phe/Tyr) and CD1 (Trp) aromatic 13C resonances can be identified in 2D 13Caro CT-HSQC and used to bootstrap resonance assignment via aromatic (H)CCH-COSY.
In rare cases when your protein does not contain Phe, Tyr, or Trp residues aromatic (H)CCH-COSY is not need. It also not suitable for assignment of HE1/CE1 resonances of His rings.
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 an aromatic 2D 13C CT-HSQC is also recommended, according to protocol HSQC_CT_13C_aro.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
- Acquisition Setup Workflow, Solution NMR Structural Biology
- PRESAT_bio.nan
- HSQC_15N.nan
- HSQC_13C.nan
- HNCO_3D.nan
- Biotop-Calibration and Acquisition setup
Create hCCH-COSY_aro 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 hCCH_COSY_aro_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 hCCH_COSY_aro_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) Load optimized parameters from BioTop. This module allows to optimize and define additional common parameters for a given dataset that can be applied to multiple experiments.
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.
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 parameters' 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 if not default. This view shows the three dimensions, F3(1H), F2(13C), and F1(13C) in columns.
Parameters to check:
- FnTYPE - 'traditional planes' or 'non-uniform sampling' ( see step 2.5 below )
- NS - minimum 2
- DS - 32-128 'dummy' scans that are not recorded (multiple of 8); allows system to reach steady state
- 3 SW - 1H spectral width (~12-15 ppm, defined in BioTop)
- 2 SW - 13Caro (INEPT) spectral width (~40 ppm )
- 1 SW - 13Caro (COSY) spectral width (~40 ppm )
- O1 - 1H H2O offset in Hz (calibrated with BioTop or calibo1p1 )
- O2P - 13Caro offset (~125 ppm, defined in bioTop )
- O3P - 15N offset, for Trp and His rings (~130 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 13Caro (INEPT) time domain real points (2 AQ ~4.5 ms for CT evolution only, or up to ~6ms for semi-CT evolution, limited by 1JCC couplings )
- 1 TD: Number of 13Caro (INEPT) time domain real points (2 AQ ~4.5 ms for CT evolution only, or up to ~6 ms for semi-CT evolution, limited by 1JCC 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 are appropriate, however it's useful to compare values in the fields against those proposed by the original parameter file and the pulseprogram.
- D1 - 1- 2 sec
- P1 - 1H 90º high power pulse (calibrated with calibo1p1 or BioTop)
- P3 - 13C 90º high power pulse (calibrated with BioTop)
- P21 - 15N 90º high power pulse (calibrated with BioTop)
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 window and set the desired NusAMOUNT [%] sampling density.
For adequate reconstruction, number of NUS points should be larger than the number of expected peaks in any of the 2D 13C-13C planes. Total number of aromatic (H)CCH-COSY peaks varies by residue type: 11 (Trp), 7 (Phe), 4 (Tyr), 2 (His, diagonal only). NUS sampling amount should thus be ~10-30% provided there is sufficient S/N.
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
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, A.U. Singer, D.R. Muhandiram & J. D. Forman-Kay, J. Magn. Reson. B 101, 333 - 337 (1993))
'nusPGSv8' written by Scott Anthony Robson 2013