France 2 - The platforms of the Instituted de Biologie Structural managed by Integrated Structural Biology Grenoble (ISBG).
Nuclear magnetic resonance (NMR) has become an important technique for the determination of the three-dimensional structure of biological macromolecules. The ability to characterize both solution structure and dynamics, possibly in the presence of physiological partners, have made NMR an essential tool for understanding biological processes. The study of molecular complexes, even in the case of weak affinity, opens up powerful opportunities for the development of pharmacologically active molecules. NMR spectroscopy provides access to a wide range of biomolecular systems in solution, in cell extracts or living cells, in lipid micelles, in a micro-crystalline environment, or in a biological membrane.
Analytical ultracentrifugation (AUC) studies macromolecules in solution that are subjected to a centrifugal field. AUC combines the separation of the macromolecules and the analysis of their transportation in view of a rigorous thermodynamics. AUC is particularly suitable for studying the interactions of membrane proteins, glycosylated proteins, complexes between proteins and polymer or RNA.
The EM platform at IBS provides access to a full range of instruments (three in total) for national and European users via FRISBI and Instruct respectively. This includes classical quality control negative staining experiment (Tecnai 12 EM) prior to setting up cryo conditions (F20 EM equipped for cryo and tomography). Data acquisition can also be carried out on the F20 EM. Our third EM : the state-of-the-art Polara EM (Tecnai F30, 200-300 kV) is equipped with a direct electron detector (K2 summit) allowing (pseudo-) atomic resolution on biological samples. The Polara is able to do single particle imaging and single axis tomo. It is also equipped with a GIF. http://www.ibs.fr/platforms/structure-determination/cryo-electron-microscopy/
ESPRIT: Screening of tens of thousands of constructs of a single gene to identify well-behaving soluble constructs.
**OVERSUBSCRIBED, LONG DELAYS EXPECTED** Please contact email@example.com before submitting an application to Instruct. The Instruct financing will cover the costs of the consumables, but the visitor will be required to contribute towards the costs of flight and accommodation. Please ask D. Hart for more details.
HTMPC platform is now part of the HTX platform (EMBL). It is fully equipped to accomplish successful crystallization of your membrane protein (in meso, bicelle, vapor diffusion). Robotic systems nanovolume dispenser (Mosquito and NT8) allows to screen 96 precipitant conditions with small amount of sample (5-10 µl per plate) in less than 10 minutes. Our platform works with commercially available kits specialized in membrane protein crystallization. An automated imaging system, RockImager 1500 (Formulatrix), visualizes crystallization drops with visible and UV lights, which allows a better identification of protein crystals. The smallest crystals which can be confidently detected are about 10 micrometers. CrystalDirect technology developed at the platform allows automatic harvesting of the crystals. LCP-FRAP robot measures the mobility of the protein in the phase and allows a pre-screening of the protein and crystallization conditions prior to crystallization.
The Cell Free expression platform of IBS is devoted to large scale production (milligrams quantities) of soluble proteins, membrane proteins and RNAs for structural studies (X-ray, NMR...). The platform benefits from scientific input of the Membrane Transporters and Membrane and Immunity teams for expression and solubilisation of membrane proteins and from the group for isotopic labelling of RNA and proteins. The Cell Free platform is setting up a quality process aimed at an ISO 9001 labelling. We propose the following services: Small scale protein expression screening for optimisation of protein constructs. Optimisation of reaction conditions for large scale expression. Small scale RNA expression screening.
The isotope labelling platform of the IBS is devoted to the large scale production of proteins uniformly or specifically enriched in stable isotopes (15N, 13C and 2H) for biomolecular NMR spectroscopy studies. The platform is offering access to innovative labelling schemes, developed locally on the platform by the IBS. The platform benefits from extensive scientific contacts with the IBS high field NMR Spectroscopy and Cell-Free platforms. Activities proposed by the platform: Users have access to the facility for large scale expression of labelled proteins in E.coli, under the supervision of a qualified platform engineer. Dedicated bench, optimised protocols and adequate isotopically labelled materials are available to users.
The RoBioMol platform, hosted by the Pneumococcus Group of the Institut de Biologie Structurale, offers high throughput molecular biology processes using automates. Current services include: - gene cloning, - site-directed mutagenesis, - expression and purification test of proteins expressed in E. coli - automated plasmid preparation. These services are available for a minimal number of cloning / expression tests of 24 samples. Data are processed and managed via a Laboratory Information Management System (RoBioLIMS) specifically developed for the platform by the CEA (GIPSE).
The RoBioMol platform, hosted by the Pneumococcus Group of the Institut de Biologie Structurale, offers high throughput molecular biology processes using automates. Current services include: - screening of detergents for membrane protein solubilization and affinity purification - detergent exchange on solubilised membrane proteins. These services are available for a minimal number of detergent screening of 24 samples.
The Biacore technology allows real-time detection and monitoring of biomolecular binding events. One of the interacting molecules (the ligand) is bound to the biosensor surface (sensor chip), whereas the other is delivered to the surface in a continuous buffer flow through a microfluidic system. Binding to the immobilized molecule is followed by surface plasmon resonance, which detects mass changes at the sensor surface. Recording SPR signal variation as a function of time (sensorgram) for several analyte concentrations allows to determine the association and dissociation rate constants, and to derive the affinity constant. This technology also allows to measure the concentration of functional molecules and the interaction stoichiometry.
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