NMRtist

NMRtist Use Cases

Written by: Piotr Klukowski
Published: July 29, 2024, 10:03 a.m.

List of the most common NMRtist use cases:

Inputs for NMRtist Applications
Automated peak picking
Automated de nbloovo chemical shift assignment
Automated de novo structure determination of protein monomer
Automated de novo chemical shift assignment and structure determination of protein-ligand complex
Manual refinement of automated chemical shift assignments and structure determination results
Automated chemical shift transfer
Automated structure based chemical shift assignment
Automated completion of incomplete manual assignment

Inputs for NMRtist Applications

The table below lists the input files that can be used with the NMRtist applications. These inputs play a role in different use case scenarios where the assistance of the platform in data analysis is possible.
By understanding the specific uses and formats of these input files, users can effectively leverage the capabilities of NMRtist to enhance their data analysis workflows.

Input file	Data types	Practical application	Example source
Peak list	Sparky (.list), XEASY (.peaks)	Provides manual peak lists as input for the NMRtist application	Manual peak picking Manually refined output of the automated peak picking
Chemical shift statistic	XEASY (*.stats)	Defines chemical shifts for the automated chemical shift transfer	Assignment of source protein that is used for transfer Chemical shift predictions (e.g. UCBShift) based on AlphaFold structure
Chemical shift list	XEASY (*.prot)	Provides partial assignment for the automated chemical shift completion	Incomplete list of manually assign chemical shifts Manually refined output of the automated chemical shift assignment
Protein structure	PDB (*.pdb)	Defines the protein fold for the automated structure-based chemical shift assignment	Protein model obtained with experimental methods In-silico prediction (e.g. AlphaFold)
Distance restraints	CYANA (.upl. .lol))	Defines distance restraints to be used in the protein structure calculation	Distance restraints obtained in experiments not reported in NMRtist project
Angle restraints	TALOS (*.aco)	Defines angle restraints to be used in the protein structure calculation	Angle restraints obtained in experiments not reported in NMRtist project

Automated peak picking

Create and open NMRtist project
Upload NMR spectra to the project storage
Run the application "ARTINA: peak picking" using the NMR spectra you would like to analyze as the input.

Automated de novo chemical shift assignment

Create and open NMRtist project
Upload NMR spectra to the project storage
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: chemical shift assignment" using NMR spectra you would like to analyze as the input.

Automated de novo structure determination of protein monomer

Create and open NMRtist project
Upload NMR spectra to the project storage
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: structure determination" using NMR spectra you would like to analyze as the input.

Automated de novo chemical shift assignment and structure determination of protein-ligand complex

Prepare CYANA library file with definition of the ligand
Create NMRtist project using CYANA sequence (*.seq) and ligand definition (*.lib). Open the project.
Upload NMR spectra to the project storage
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: chemical shift assignment" or "ARTINA: structure determination" using NMR spectra you would like to analyze as the input.

Automated chemical shift transfer

Chemical shift transfer allows finding the unknown assignment of the target protein, given a limited set of spectra of the target protein and the known assignment of the source protein.
The chemical shifts of the source and target proteins must be similar, but not necessarily identical.

Create and open NMRtist project
Upload NMR spectra of target protein to the project storage
Prepare and upload *.stat file with chemical shifts and their tolerances of the source protein. Example *.stat file is available in user manual
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: chemical shift assignment" using NMR spectra and *.stat file as the input. NMRtist will automatically recognize *.stat file in the input and trigger the chemical shift transfer.

Automated structure based chemical shift assignment

Structure-based chemical shift assignment assigns the chemical shifts of a protein given its NMR spectra and protein structure.
This use case is designed for NMR studies, where the fold of the protein is known. Alternatively, one can use AlphaFold prediction as the input, if experimental structure (e.g. X-ray) is not available.
An extensive evaluation of this procedure is available in our manuscript (doi/full/10.1126/sciadv.adi9323).

Create and open NMRtist project
Upload NMR spectra of target protein to the project storage
Upload *.pdb file with structure model to be used for structure based assignment
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: chemical shift assignment" using NMR spectra and *.pdb file as the input. NMRtist will automatically recognize *.pdb file in the input and trigger the structure-based assignment.

Automated completion of incomplete manual assignment

This use case is applicable when a partial assignment (e.g., backbone) is available from previous studies, and the objective is to find the complete assignment given newly recorded spectra (i.e., backbone and side-chain).

Create and open NMRtist project
Upload NMR spectra of target protein to the project storage
Prepare and upload *.prot file with known chemical shifts. Example is available in user manual
Run application "ARTINA: peak picking" using NMR spectra you would like to analyze as the input.
Verify that the output of the automated peak picking does not contain any anomalies, especially those related to spectra referencing.
Run application "ARTINA: chemical shift assignment" using NMR spectra and *.prot file as the input. NMRtist will automatically recognize *.prot file in the input and trigger the chemical shift completion.

The results obtained through NMRtist automated data analysis can be refined manually using the "human-in-the-loop" procedure.

Analyze results of the NMRtist job and identify the bottleneck. For example, if you find atoms with weak assignments in the protein backbone, the problem might be linked to insufficient number of peaks identified in the backbone spectra. If you identify that number of NOEs is insufficient, the problem might be linked to insufficient number of cross-peaks detected in the NOESY spectra.
Manually refine the identified bottleneck. For example, if the problem is linked to an insufficient number of cross-peaks detected in the C13NOESY spectrum, download the C13NOESY peak list from the NMRtist results, open it in dedicated NMR software (e.g., Sparky, CCPN), and refine by removing wrongly identified peaks (false positive predictions) and adding missing peaks (false negative predictions).
Upload the refined file to your NMRtist project
Use the refined file as input for the application call. Note: If you provide a refined peak list, use it instead of the corresponding NMR spectrum.

Frequently Asked Questions

How do I handle sequences with custom residues?
To work with custom residues, you can define them in a CYANA library file and include them in your sequence file. These custom library and sequence files can then be used to create a new project. Once the project is created, handling custom sequences is the same as working with standard amino acid sequences. During runtime, NMRtist applications automatically recognize and handle custom residues.

How do I handle disulfide bridges?
For cysteines involved in forming disulfide bridges, mark them as CYSS in the protein sequence. If you have information about specific pairs of cysteines that form bridges, this can be provided in a *.upl file.