The Institute of Crystallography (IC-CNR) carries out basic and applied studies and research activities in various fields of science. They range from the development of crystallographic methodologies and automatic calculations of X-ray diffraction to chemistry and structural biology.
The IC-CNR structural biology groups are strongly focused on the structure-activity-function relationship in proteins relevant to biomedicine and biotechnology. The aim is the understanding of the molecular determinants underlying their biological activities as well as the rational design of new drugs and diagnostics. Activities range from the study of proteins relevant as potential drugs against neurodegenerative disorders, to the study of proteins of critical importance for cancer and, to the investigation of proteins that are crucial for the virulence of bacterial pathogens.
The high-throughput crystallization screening facility is shared with the Structural Biology Laboratory at Elettra Sincrotrone Trieste.
Platforms available for access through Instruct-ITALIA
- X-ray macromolecular crystallography
- Small Angle X-ray Scattering (SAXS)
- High-Throughput Screenining facility
- Molecular Biophysics
IC-CNR together with Elettra Sincrotrone Trieste manages the XRD-1 variable energy beamline. The XRD-1 is dedicated to high throughput X-ray macromolecular crystallography experiments. Tunable energy range for SAD/MAD experiments, automated sample mounting in a cryogenic environment and high speed large area detector are some of the important features of this beamline.
Details on the SAXS/WAXS instrumentation available at XMI-L@b and on specific instrumentation for molecular biophysics can be found below.
X-ray macromolecular crystallography
The platform have the access to the X-Ray Diffraction 1 Beamline @ELETTRA.
The X-Ray Diffraction 1 (XRD1) beamline, a partnership between IC-CNR and ELETTRA, has been designed primarily for macromolecular X-ray crystallography.
The insertion device of the XRD beamline is a multipole wiggler (1.607 T), with a useful energy range from 4 to 21 keV (photon flux 1012-1013 ph/s).
The optics consist of a vertical collimating cylindrical mirror, a double-crystal Si(111) monochromator followed by a bendable focusing toroidal mirror. The multipole wiggler spectrum includes high photon flux at low energies, allowing the optimization of the anomalous signal of several heavy atoms and offering the enhancement of the sulfur anomalous signal. The energy resolution is 10-4 for routine and MAD experiments. XRD-1 beam is focused at 700 x 200 μm² FWHM (precision slits for beam shaping) at sample.
The experimental setup consists in: a Huber goniometer with κ geometry fully controllable from remote; an Oxford Cryostream 700 (temperature range 80 – 400 K); an in house developed sample changer handling only SPINE standard sample holders and vials stored into cryo-vial baskets (5 baskets, i.e. up to 50 samples) loaded into the dispensing dewar; an Amptek X-123 SSD X-ray fluorescence detector; a Pilatus 2M (Dectris) detector, with an active area of 254 x 289 mm2 (1475 x 1679 pixels, which are 172 x 172 µm2 in size). The readout time is 3.6 ms and data collection in shutterless mode allows a complete dataset to be collected in few minutes; an Xcell (Oxford cryosystems) for Xe/Kr pressure protein crystals derivatization.
Remote data collection is available.
Software data processing: ADXV, iMOSFLM, XDS, Xia2
Software data analysis: CCP4, CNS, Coot, HKL2MAP, Il Milione, Phenix, SHELX, Solve/Resolv
Small Angle X-ray Scattering (SAXS)
Small and Wide Angle X-ray scattering in transmission (SAXS and WAXS) can provide a large amount of structural and morphological information: the crystalline atomic order can be mainly studied by WAXS, the nano-structure/morphology can be assessed by SAXS. Simultaneous acquisition allows relating crystallinity occurring at different scales. A micro-source together with a scanning acquisition mode allows transforming a series of raw data into a quantitative microscopy, as offered by the X-ray Micro-Imaging Laboratory (XMI-L@b) which has the proper instrumentation and has developed in-house the software needed for data collection and analysis.
The X-ray Micro Imaging Laboratory (XMI-LAB) is equipped with a Fr-E + Super-Bright (Rigaku) rotating copper anode micro-source (λ = 0.154 nm, 2475 W) coupled through a focusing multilayer optics Confocal Max-Flux to a SAXS/WAXS three pinholes camera equipped for X-ray scanning microscopy. An image plate (IP) detector (250 × 160 mm2, with 100 μm effective pixel size), with an off-line RAXIA reader, is employed to collect WAXS data. A Triton 20 gas-filled proportional counter (1024 x 1024 array, 195 mm pixel size) is used for SAXS acquisition. The spot size at the sample position can be reduced to a minimum of 70 x 70 μm2.
High-Throughput Screening facility
The automated platform is optimized for the high-throughput crystallization of macromolecular samples, in order to maximize the chances of crystallization. A Thermo Scientific Matrix Hydra II eDrop liquid dispenser system is equipped with a motorized XYZ-platform, with 96 precision stainless steel syringes and an additional high-precision noncontact single-channel microsolenoid dispenser, which transfers 100nl-50ml of protein solution. Up to 100µl of premixed cocktails can be aspirated with the 96-syringe-assembly and dispensed into reservoir and droplet wells within 60s. Setup for crystallization trials: vapor diffusion - sitting drop or microbatch. Temperature controlled chambers (KW Apparecchi Scientifici) at 21° C and 4° C. A Tecan liquid handler (Freedom EVO 150) to set up crystallization screens and a Mosquito (TTP Lab Tech) crystallization robot (Accessible at Elettra’s BioLab).
Grating-Coupled Interferometry (GCI) is a method based on waveguide interferometry to monitor and characterize molecular interactions and to determine binding kinetic rates and affinity constants. Waveguide Interferometry like other optical label-free methods, such as Surface Plasmon Resonance (SPR), detects refractive index changes within an evanescent field near a sensor surface due to changes in mass by complex formation of the interacting molecules.
In contrast to Surface Plasmon Resonance where the surface plasmon is quickly attenuated by the thin gold film, the light in waveguide interferometry can travel over the entire interaction length. Hence, more binding events contribute to the signal and an intrinsically higher primary sensitivity is achieved making waveguide interferometry the most sensitive optical principle for label-free interaction analysis. Furthermore, the evanescent field penetrates less deep into the sample and less disturbance by bulk refractive index changes is experienced.
With its unrivaled flexibility and leading sensitivity across the widest application range, GCI brings all the benefits of label-free analysis to applications including fragment-based drug discovery, kinetics of weak binders with fast off-rates, analysis of complex analytes (i.e. serum) and antibody characterization.
WAVE – Creoptix Bionsensors
Innovative combination of microfluidics and sensor in a single disposable cartridge.
Variety of chip are available in a broad range of surfaces (–NH2, -SH, -CHO, -OH or –COOH; NTA for capturing His tagged molecules; streptavidin for capturing biotinylated molecules).
Fully enclosed, the sensor surface is protected from contamination or damage. Unique, no-clog microfluidics design minimizes downtime.
Ultra-fast transition times of 150 msec; Reliable determination of off-rates of 5 sec–1 and faster, enabling off-rate screening of weakly binding analytes; Stable multi-hour dissociation analysis for high-affinity binders.
The software allows Import and export of the data to and from Excel and other formats. It is capable of using predefined or arbitrary customized evaluation models for kinetic fit. It quickly generates reports and editable Word files through an automated report generator.
Isothermal Titration Calorimetry
Isothermal Titration Calorimetry (ITC) is a long-established technique that has many applications in drug discovery and development and increasingly in understanding the biological pathways that allows identification of drug targets.
ITC has become one of the mainstays to directly characterize the thermodynamics of a wide range of bio-molecular interactions: antibody-antigen, protein-protein, protein-ligand, DNA-ligand and RNA macromolecule as well as the binding kinetics of enzyme-catalyzed reactions.
In structural and functional biology, two of the most important questions are: i) how tightly does a small molecule bind to a specific interaction site? and ii) how fast does the reaction take place if the molecule is a substrate and is converted to a product?
ITC is a quantitative technique that can determine the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution.
Modification (derivatisation with fluorescent probes) of the binding partners and/or immobilization and/or chemical modification are not required for data acquisition.
When combined with structural information, ITC data provide deeper insights into structure–function relationships and the mechanisms of binding. This is especially important when rationally improving the binding affinity of drug-related molecules towards a protein target of pharmacological interest.
The system has a 200 μl coin-shaped non-reactive (Hastelloy) sample cell and an automated injection syringe (40 μl volume; smallest injection volume: 0.1µL) and provides direct measurement of the heat exchanged because of mixing precise amounts of reactants. Data analysis is performed using Origin™ software, wherein the user obtains the stoichiometry (n), dissociation constant (KD), and enthalpy (ΔH) of the interaction. The Origin software can also be used to fit models that are more complicated.
Dynamic Light Scattering
Dynamic light scattering (DLS), also known as photon correlation spectroscopy, measures time-dependent fluctuations in the scattering intensity arising from particles undergoing random Brownian motion. Diffusion coefficient and particle size information can be obtained from the analysis of these fluctuations. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of macromolecules. DLS provides the ability to study the homogeneity of proteins, nucleic acids, and complexes of protein–protein or protein–nucleic acid preparations, as well as to study protein–small molecule interactions.
Predisposition of proteins to crystallization correlates with the homogeneity of the molecules in solution. DLS is particularly well suited for evaluating protein homogeneity (degree of polydispersity) under multiple conditions and at concentrations commensurate with crystallization conditions.
Protein Solutions DynaPro MSX Dynamic Light Scattering System
Specifications: Size range (Radius - nm): 0.5 to 1000; Minimum Concentration: 0.1 mg/ml; Laser wavelength: 830 nm; Laser power: 0 to 50 mW; Minimum sample volumes: 2 µl and 12 µl quartz cells, or 50 µl UVette Eppendorf Cuvette with adapter (2mm or 10mm optical path length).
Surface Plasmon Resonance (SPR)
SPR Technology is a surface-sensitive analytical method for chemical and biochemical sensing that detects changes in the refractive index in the immediate vicinity of the surface layer of a sensor chip. While there are many biosensing instruments available, SPR technology makes observing binding behavior on a molecular level easier and more accurate than ever before. THE SENSÍQ PIONEER AE platform can work to study biomolecular interactions via label-free techniques. The SensÍQ Pioneer offers exceptional data quality and sensitivity in a fully automated, affordable surface plasmon resonance system. Whether your research involves small molecule drug discovery, antibody selection and screening or rigorous protein-protein interaction kinetics studies, the flexibility and straightforward approach to SPR analysis of the SensÍQ Pioneer can help solve your most difficult problems
Pioneer AE ( SensiQ)
SAXS is a powerful method to analyze proteins and other macromolecules in solution, and for understanding structure and association under physiological conditions. It can give insights on the effects of buffer composition, the quaternary state of proteins as well as their oligomerization behavior, assembly, folding and their interactions with other macromolecules or small ligands.
IC-CNR offers a wide range of expertise and skills in sample preparation, BioSAXS and SEC-BioSAXS measurements at high brilliance synchrotron facilities (ESRF-Grenoble, DIAMOND-Oxford, and PETRA III-Hamburg), data processing, analysis and model building.
Software for data processing and analysis: ATSAS, developed in Dmitri Svergun's group at EMBL Hamburg, is the world most comprehensive and powerful program suite for small angle scattering data analysis from biological macromolecules in solution.
ATSA can evaluate the overall geometrical parameters of the sample (radius of gyration, molecular weight etc.); reconstruct the shape of proteins and other macromolecules in solution ab initio and build models using data from complementary methods (X-ray crystallography, NMR, bioinformatics etc.); analyze oligomeric mixtures and flexible systems (including intrinsically disordered proteins).
Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques. Briefly, circular dichroism is defined as the unequal absorption of left-handed and right-handed circularly polarized light with data collected at ~190 to 250 nm (far UV) and from 250 to 300 nm (near UV). Because the spectra of proteins are so dependent on their conformation, CD can be used to estimate the structure of unknown proteins and monitor conformational changes due to temperature, mutations, heat, denaturants or binding interactions.
In addition to the intrinsic circular dichroism of the protein backbone, when ligands with chromophores bind to proteins they may develop strong extrinsic CD bands that can also be used to follow binding.
The aromatic chromophores of proteins, which have bands in the near ultraviolet region, are often in asymmetric environments and can be used to examine whether mutations change the tertiary structure of proteins. While CD does not give the secondary structure of specific residues, as do X-ray crystallographic and NMR structural determinations, the method has the advantage that data can be collected and analyzed in a few hours on solutions of samples containing 20 µg or less of protein in aqueous buffers under physiological conditions.
JASCO Spectropolarimeter J-810 (Accessible at Elettra’s BioLab)
The system will accept standard 1cm x 1cm quartz cuvettes. A Spacer is included to accommodate smaller cuvettes: demountable cells (0.10 mm optical path length – volume ~60 µL); quartz cells (1 mm or 2mm path length – volumes ~250 µL, ~700 µL).
Differential Scanning Fluorimetry
Differential Scanning Fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become an important label-free technique for biophysical ligand screening and protein engineering. This method makes use of a fluorescent dye – typically SYPRO Orange or ANS – that is quenched in an aqueous environment but becomes strongly fluorescent when bound to exposed hydrophobic groups of a protein. By heating one’s protein of interest in the presence of such a dye, the thermal unfolding transition can be monitored spectrophotometrically. Because ligands that interact with proteins typically stabilize the folded protein, this leads to a positive shift in the midpoint of the unfolding transition i.e. the melting temperature, Tm.
DSF has also become a commonly used approach for detecting protein-ligand interactions, particularly in the context of fragment screening. Upon binding to a folded protein, most ligands stabilize the protein; thus, observing an increase in the temperature at which the protein unfolds as a function of ligand concentration can serve as evidence of a direct interaction.
BioRad CFX96 qPCR (Accessible at Elettra’s BioLab), 96-well plate format using 25µL reaction volumes; Heating and cooling method: Peltier; Temperature range: 0-100°C; Range of excitation/ emission wavelengths: 450-730 nm.