Considerations for NMR spectroscopy structure determination experiments

The following points are guidelines for protein NMR experiments. Many applications of NMR spectroscopy work around these conditions. Please inquire for advice on your particular project.

In NMR spectroscopy, concentration is proportional to signal, so the higher the concentration the better the data acquired. For a peptide sample, concentrations of 2-5 mM are usually possible, while the limit is lower for a larger protein. However, a high concentration is not always optimal for systems which form dimers or larger size oligomers.

Labeling or not?
The amount of labelling required is dependent on the peptide/protein size and concentration possible. For peptides and proteins consisting of up to 40 residues, no labelling is strictly necessary since 15N and 13C natural abundance spectra are possible with modern high-field spectrometers equipped with cryo-probes. However, labelling is beneficial, especially if the concentration is low, and 15N-isotope labelling is usually sufficient.
For proteins above 40 residues, 15N and 13C-labelling is essential, mainly due to spectral overlap in 2D-spectra, where 15N and 13C-labelling allows for the acquisition of 3D-NMR spectra and reduced spectral overlap. 15N and 13C-labelling is in most cases easily done by expression in E. coli using minimal media, where especially the cost of the 15N-isotope labelling is low. For proteins in the range 20-30 kDa and above, 2H isotope labelling will increase signal to noise and is often required for successful assignment and structure determination.

• Size?
For high-resolution tertiary protein structure determination by NMR spectroscopy, the molecular weight of the protein is typically less than 30 kDa. Larger proteins (up to 100 kDa in favorable cases) can be studied in projects that aim at identifying ligand binding sites or characterizing the physical state of the construct.

• Amount?
As a rule of thumb, the typical protein NMR sample should have a concentration of 0.5-1.0 mM in 500 µl, corresponding to 5-10 mg protein for a 20 kDa protein.
Peptide samples for NMR are preferably run at higher concentrations, where concentrations of 1-5 mM correspond to 1.5-7.5 mg peptide.

• Stability?
For structure determinations of proteins, the protein needs to be stable at room temperature for at least a week. However, this is easily achievable using standard protein purification methods and optimization of solvent conditions.
For structure determination of peptides, the peptide needs to be stable in solution for 2-4 days.

• Non-natural amino acids?
Structure determination of peptides with non-natural amino acids is typically no problem, although some considerations have to be made with regard to peak assignment and parametrization of the amino acids for structure determination

Bruker Avance III HD spectrometer
Bruker Avance III HD spectrometer

We generally run peptide/protein NMR structure determination projects as two work packages as outlined below:

Work Package 1: Assignment and feasibility
In order to assess whether it is possible to determine the structure of a peptide/protein, a standard set of NMR experiments is acquired. For a peptide, this generally consists of standard 2D NMR experiments to verify the primary structure as well as a secondary structure estimation using the acquired spectra. Typically all the available 1H and selected 13C and 15N resonances are assigned. For a protein, a set of standard 3D NMR experiments is acquired in order to assign backbone resonances, where generally >95% of the available resonances can be assigned. The outcome of this work package results in assignment of the primary structure, a secondary structure estimation and whether it is feasible to do a structure determination of the peptide/protein.

Work Package 2: Structure determination using restrained molecular dynamics simulations
For a peptide, in general all the structural restraints can be generated from the experiments in work package 1 and therefore this step entails iterative structure calculations in order to achieve a peptide structure ensemble consistent with the NMR restraints. For a protein, full assignment of available resonances is performed using 3D NMR experiments, followed by the generation of structural restraints and iterative structure calculations in order to achieve a protein structure ensemble consistent with the NMR restraints. The outcome of this work package is a protein structure ensemble showing the structures most consistent with the structural restraints.

Further work packages can be designed based on the customer’s needs, such as titration experiments to identify binding epitopes, filtered NOE experiments or transfer NOEs to determine compound-protein complexes, relaxation measurements or residual dipolar coupling measurements to study protein dynamics.

Oxford 800 magnet, Bruker Avance III HD spectrometer
Bruker 600 NMR spectrometer

For high throughput NMR spectroscopy data acquisition we have access to a number of ultra high magnetic filed NMR spectrometers:

900 MHz Bruker Avance III HD

4 RF channels
Triple-axis pulsed field gradients
TCI cryoprobe (
1H/13C/15N, 5 mm)
Triple resonance probe (1H/13C/15N)

800 MHz Bruker Avance III HD

4 RF channels
Triple-axis pulsed field gradients
TCI cryoprobe (
1H/13C/15N, 3 mm)

800 MHz Bruker Avance III HD

4 RF channels
Triple-axis pulsed field gradients
TXO cryoprobe (
1H/13C/15N, 5 mm)
Triple resonance probe, 8 mm

700 MHz , Bruker Avance III spectrometer

4 channels 1H/19F/13C/15N
5 mm QCI cryoprobe

3x600 MHz Varian Inova/Bruker

3-4 RF channels
Triple-axis pulsed field gradients
Tripple resonance cryo-probe (
Triple resonance probe (
4 mm NANO probe
Diffusion probe (

For more details on NMR spectroscopy services please view our services page.

Custom NMR spectroscopy services provided by SARomics Biostructures include:

Peptide or protein assignment and secondary structure estimation
Peptide or protein 3D structure determination
Protein-ligand & protein-protein complex structure determination
Fragment screening using 1D or 2D NMR experiments
Epitope mapping and dissociation constant (Kd) determinations for compounds
Higher order structure (HOS) studies of biosimilars, comparison with originator antibody and formulation optimizations
Expression and purification of 2H, 15N and/or 13C-labelled protein in E.coli for protein NMR studies

The price of our NMR spectroscopy services is highly competitive!