Institution: IC
Departement: DSCTM
Typology: Academic collaboration, Facility, Skill, Software
Location: Via Amendola 122/o, 70126, Bari, Italy
Contacts:
- Benny Danilo Belviso (danilo.belviso@cnr.it)
- Anna Giovanna Sciancalepore (annagiovanna.sciancalepore@cnr.it)
Description:
Our expertise lies in purification and advanced characterization by X-ray-based techniques of bio-macromolecules to perform cutting-edge structural biology investigations on proteins and their complexes. Our proven experience enables us to handle challenging systems such as membrane proteins and protein-ligand complexes. Regarding protein-ligand adducts for crystallographic studies, in addition to standard soaking experiments using single ligands, we also perform fragment screening for rational drug design. We offer the opportunity to perform structural biology studies by X-ray diffraction for high-resolution structure determination and provide established proficiency to obtain crystal structure information about protein-ligand interactions. Moreover, we are experts in Bio Small-Angle X-ray Scattering (BioSAXS) and Bio X-ray Absorption Spectroscopy (BioXAS) data collection and analysis. Our strength lies in our ability to handle difficult-case data, thanks to the in-depth knowledge of structural solution methods within our team and the capability to develop ad-hoc analysis procedures. Our instrumentation includes a lab-scale purification chromatographic system and an Oryx4 liquid handler robot, for crystallization experiments in various setups, microseeding, and Lipidic Cubic Phase (LCP). We introduced advanced methods to increase crystallization chances of proteins by using polymeric membranes, hydrogels, and Deep Eutectic Solvents (DES) [10.1038/s41596-023-00869-w]. Skills: Purification of bio-macromolecules, bio-macromolecule complexes, and protein-ligand adducts by chromatographic techniques (affinity, IEX, SEC) with CiPP (Capture, intermediate Purification, Polishing) strategy Crystallization of bio-macromolecules, bio-macromolecule complexes, protein-ligand adducts, including membrane proteins Fragment screening experiments for rational drug design X-ray diffraction data collection and structural solution including data analysis for protein-interacting ligands identification BioSAXS data collection and structural solution BioXAS data collection and analysis for metal-ion coordination-shell identification Facility: 2Cryst-Lab BioCryst-Lab Main instrumentation: NGCQUEST 10+: Chromatographic system for lab-scale purification applications. 2x10 ml/min pumps, accurate gradients for high-resolution separations, multi-wavelength detector for simultaneous detection of proteins, peptides, and nucleic acids. Equipped with chromatographic columns for affinity (His-trap FF), IEX (Hitrap Q/SP FF), and SEC (S70, S200GL, Enrich650) separations. NanoDrop 2000c: Nanodrop for high absorbance accuracy (3% at 0.74 Abs at 350 nm) spectrophotometric measurements, wavelength range 190–840 nm, sample volume 0.5–1000 µL. Oryx4: Liquid handler robot for crystallization experiments in microbatch, sitting and hanging drop setup. Enables LCP, optimization (2D and cross-matrix), microseeding, and additive optimization experiments. Volume of droplets ranges from 0.1 to 8.0 µL. Xtal SpectroLight 600: Benchtop instrument for particle sizing by DLS in small sample volumes (0.08–1 µL). The sample carrier is a standard 96-well plate. It can be used for assessing sample quality and stability before crystallization procedures, SAXS, or Cryo-EM measurements. Software: XDS Dials ccp4 Phenix SIR GlobalPhasing PanDAA ScÅtter ATSAS Athena Artemis Academic collaborations: Dipartimento di Farmacia - Scienze del Farmaco (UNIBA) Dipartimento di Bioscienze, Biotecnologie e Ambiente (UNIBA) Dipartimento di Chimica (UNIBA) ISPA (CNR) Nanotec (CNR) IPSP (CNR) IPCF (CNR) STIIMA (CNR) ITM (CNR) Dip. di Chimica e Biologia (UNISA) Dip. di Scienze Chimiche (UNINA) Lab. Materiali Ceramici e Compositi per l’Industria Manifatturiera (ENEA) Diamond Ltd Synchrotron Technical University of Munich Dipartimento di Neuroscienze, Biomedicina e Movimento (UNIVR)
Team:
- Benny Danilo Belviso (bennydanilo.belviso@cnr.it)
- Anna Giovanna Sciancalepore (annagiovanna.sciancalepore@cnr.it)
- Vincenzo Mangini (vincenzo.mangini@cnr.it)
- Rocco Caliandro (rocco.caliandro@cnr.it)
- Daniela Malerba (daniela.malerba@cnr.it)
Fig.1 The three-stage purification strategy Fig.2 NGC QUEST10+ chromatographic system Fig.3 Oryx4 (Liquid handler for macromolecule crystallization experiments) Fig.4 Structural solution workflow from X-ray diffraction data