Service / Technology Instance

About Solution NMR at CERM/CIRMMP in Florence, Italy

CERM/CIRMMP offers unique research capabilities in the field of solution NMR of biomolecules providing state of the art instrumentation and expertise to perform, at the highest level, the most comprehensive array of experiments needed for the structure and dynamic characterization of biological macromolecules and their complexes in solution. To attain fundamental molecular level information on cellular processes, all the standard pulse sequences for spectroscopic, structural and dynamical characterization are available. We develop tailored pulse sequences for structural determination of high molecular weight proteins and paramagnetic systems, 13C direct detection protocols for “protonless” NMR experiments, fast in-cell NMR experiments.

View All Solution NMR at Instruct

Instruments Available:

The infrastructure is equipped with high field spectrometers equipped with cryoprobes (950 MHz TCI cryo, 900 MHz TCI cryo, 700 MHz TCI cryo, 700 MHz TXO cryo, 500 MHz TCI cryo), heteronuclear detection probes at 600 MHz for low frequency nuclei to meet the most disparate experimental needs, liquid handler system and autosamplers for metabonomics and ligand screening at 400, 600 and 700 MHz. Special accessories such as high power probes (short hard pulse) at 400 and 600 MHz for paramagnetic NMR. All NMR instruments are state-of-the-art digital spectrometers of the Bruker Avance NEO/III series.

User Guide

Solution NMR platform description

The solution NMR platform includes a number of spectrometers, operating at 950 MHz, 900 MHz, 800 MHz, 700MHz, 600MHz, and 500 MHz spectrometers. All of the high field NMR instruments are state-of-the-art digital spectrometers of the Bruker Avance series. Each instrument is equipped with several probes to meet all conceivable experimental conditions. Six spectrometers are equipped with CryoProbes and two of them equipped with Automatic Sample Changer to easily and effectively perform automatic (continuous) 1D and 2D NMR experiments for library screening. One of the three 700 MHz is equipped with a TXO CryoProbe specifically designed for 13C-direct detection NMR experiments with 1H, 2H, 13C and 15N decoupling capabilities. The 800 MHz has a 4 channel probe (with XYZ gradients) and there are two high-power probes for the study of fast-relaxing systems. Additional probes to cover a wide range of frequencies, including very low-frequency nuclei and several metal nuclei, such as 57Fe, are also available.

Users are provided with access to the instrumentation and expertise to perform the most comprehensive array of experiments needed for the structure and dynamic characterization of biological macromolecules and their complexes. These services will allow users to attain fundamental molecular level information on cellular pathways which represent targets of interest in human health and diseases. Translational research will be covered by the infrastructure as described below:

NMR of proteins/protein complexes in solution: CERM/CIRMMP offers unique research capabilities in the field of high-resolution NMR of proteins, with a special expertise in metalloproteins, for i) solution structure determination of biomolecules and of their complexes, iii) investigation of protein-drug interactions, iv) methodological advancements exploiting the special properties of large and/or paramagnetic biomolecules and v) study of immobilized macromolecules.

In-cell NMR: In-cell NMR allows the characterization of biomolecules in living cells at atomic resolution. Investigation of protein dynamics, folding, conformational changes induced by binding events, posttranslational modification in the complex native environments, as well as in vivo drug screening, even de novo 3D protein structure determination are some of the aspects which can be addressed in living cells through in cell NMR. This technique is made available for both in bacterial and human cell studies, by the CERM/CIRMMP infrastructure to the users. In-cell NMR protocols and experiments for the investigation of biological macromolecules in their physiological context are now available in the Infrastructure.

Structural Vaccinology: Structural vaccinology combined with human immunology are rapidly emerging as a powerful alternative strategy for the rational design of engineered vaccines bearing multiple antigenic epitopes offering the opportunity of developing broadly effective immunity. This innovative structurally-based approach has been already successfully applied to generate an engineered single antigen that induces immunity against all known sequence variants of N. meningitides. Structural vaccinology could represent today a valid revolutionary alternative leading in the next years to an efficacious vaccine design. This approach and will be offered by CERM/CIRMMP infrastructure.

Drug discovery: Nuclear magnetic resonance (NMR) spectroscopy is a very valuable method for fragment-based drug discovery and a tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. The laboratory is fully equipped has the expertise to provide access to users interested in discovering novel therapeutics.

Direct 13C detection in solution: CERM has pioneered 13C-detection experiments to obtain the assignment of nuclei in large molecules and biomolecular complexes. These heteronuclear direct detection experiments allow the study of large macromolecules as well as highly exchangeable/mobile or unfolded systems. The technique is particularly suitable to investigate intrinsically disordered proteins able to participate in multiple interactions and to fold at binding in a template-dependent manner. These properties make them indeed commonly involved in the pathogenesis of numerous human diseases and oncogenesis, suggesting that these proteins should be seriously considered as target of great biomedical interest.

Experimental requirements

To get structural information on proteins by solution NMR, isotopically labelled samples (usually 15N and/or 15N/13C) are required. For proteins with MW > 20 kDa, deuteration is also needed. In general, the degree of deuteration required is largely dependent on spectral quality, the size and modularity of the system studied and the types of experiment performed. However, very high levels of deuteration (>90 %) are usually required for proteins with molecular weights of the order of 35 kDa or greater.

The protein sample is usually dissolved in an aqueous buffer (typically ranging from 10 to 50 mM buffer concentration) to avoid pH variations in the biological samples, which can otherwise drastically change chemical shifts during NMR spectra acquisition. Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability and avoid the following issues: slow precipitation; mixture of folded and unfolded protein; aggregation problems; degradation. A screening with several buffers (with different pHs and containing chemicals such as detergents, protease inhibitor cocktails, arginine etc.) is therefore recommended to optimize NMR samples. These buffers usually contain salts (i.e. NaCl or KCl) which often increase protein solubility. Samples high in salt or ionic strength may however yield reduced signal to noise when using cold NMR probes. The use of 3 mm tubes in this NMR probe, instead of the standard 5 mm tube, can alleviate this problem. When available, use of deuterated buffer is preferred.

Samples should be clear of precipitate and particulates and therefore need to be filtered or centrifugated in this circumstance. Samples require deuterium solvent for a lock signal and therefore water protein samples contain approximately 5-10% D2O.

Samples should be slowly transferred into and out of NMR tubes using long pipettes that reach the bottom of the NMR tube to avoid losing liquid to the walls of the tube or adding air bubbles to the sample which can negatively affect the shimming profile.

Standard NMR tubes are 5mm, which should contain at least 500 microliters of protein sample. If a small volume is required, Shigemi tubes, 5mm, use volumes of 270-300 microliters and are made of glass that is matched to the magnetic susceptibility of the solvent to be used. In the high-field cold probes, Shigemi tubes should also be used for more efficient water suppression.

A concentrated solution of a biological macromolecule at high temperature and around neutral pH represents an ideal growth medium for bacteria. Sodium azide and fluoride at less than 50 mM can be good growth inhibitors. Metal chelators (EDTA or EGTA) can also be good suppressor of microbial growth.

Structure determination by NMR typically requires a protein concentration of 0.5 mM or greater, stable for several days at the desired temperature. NMR studies for ligand binding or protein-protein interaction studies require concentrations of at least 0.05 mM.

The acquisition of standard NMR spectra takes from a few minutes (1D spectra) to a maximum of 3/4 days (3D or 4D spectra) for each experiment. A full set of experiments for protein structure determination usually takes about 15-20 days. In the case of relatively small proteins, recent advanced multidimensional data acquisition schemes have been successfully used to reduce experimental acquisition time by up to an order of magnitude or more.

The first NMR experiments acquired on site will be focused on the investigation of protein folding state of user's sample as well as its aggregation state at the selected NMR protein concentration, acquiring 1D 1H and/or 2D 15N HSQC spectra and 15N backbone relaxation properties. This will take about 5-6 days. These preliminary data will allow to evaluate the time and the experimental conditions (i.e. kind of labelled nuclei, optimal protein, buffer concentration and pH) needed to obtain a high resolution structural determination on the user's protein. A large number of pulse sequences is routinely available for assignment and structural characterization, and local and global dynamics can be easily estimated.

Instruct Centre

Instruct Centre IT


Via Sacconi 6

50019 Sesto



Solution NMR at CERM/CIRMMP in Florence, Italy


Simone Ciofi-Baffoni
Simone Ciofi-Baffoni
Login to contact
Francesca Morelli
Francesca Morelli
Login to contact