Strasbourg provides an integrated environment for structural studies of protein and complexes from sample preparation to structure determination.
The center offers access for three-dimensional structure determination using cryo-electron microscopy (cryo-EM).The centre has expertise in all aspects of single particle analysis from the automated acquisition of large data sets to image analysis . Access to the following instrumentation is provided: The Tecnai F30 Polara cryo electron microscope allows high-resolution SPA and CET automated data collection. The Titan Krios cryo electron microscope with a CMOS (FEI FALCON 4K*4K) high-sensitivity direct electron detector camera. The Tecnai F20 is equipped with a FEG operated at 100keV or 200keV.
Analytical ultracentrifugation (AUC) is a powerful technique for the characterisation of macromolecules and macromolecular self- and hetero-association processes in solution. Two types of complementary experiments, sedimentation velocity and sedimentation equilibrium, can be performed in an analytical ultracentrifuge which is a high-speed centrifuge equipped with an optical detection system. The observation of macromolecules or macromolecular complexes sedimentation gives access to their hydrodynamic and thermodynamic properties, including their size, shape, molar mass, degree of heterogeneity, oligomeric http://lbgs.u-strasbg.fr/sbgp/Public/Equipments/PurifEquip/Purif.html
IGBMC Strasbourg bacterial expression platform combines (i) a large set of expression vectors for Escherichia coli and (ii) robotized protocols, which enable a broad screening of soluble protein expression through the use of multiple fusion proteins and various growth conditions.IGBMC Strasbourg bacterial platform has also a long standing and unique expertise in protein complexes reconstitution and production by multi-expression in Escherichia coli. This technology is fully compatible with the automated protocols used for single expression.All bacterial expression vectors provided by the platform are compatible with major cloning strategies (e.g. restriction, Gateway, SLIC).
The insect cells expression platform at IGBMCThis facility is dedicated to protein and multi-protein complex production in insect cells using the baculovirus expression system for structural biology as well as for functional studies. It includes streamlined procedures for (i) the generation of recombinant baculoviruses (transfections, co-transfections) and amplification of a high titer viral stock, (ii) evaluation of recombinant baculoviruses and expression optimization using adherent cells or suspensions and (iii) protein expression in various scales and formats including production of multi-protein complexes.
IGBMC-CERBM Strasbourg provides expression in mammalian cells.Over expression of proteins in mammalian cells (hamster BHK21 cells) is achieved using an attenuated vaccinia virus vector (Modified Vaccinia Virus Ankara-MVA) that is user safe and may be handled under BSL1 conditions. Genes of interest are cloned downstream of a bacteriophage T7 promoter in a first step to check for feasibility. Protein expression is then examined after plasmid transfection into BHK21 cells and co-infection with a specialized viral vector encoding the bacteriophage T7 RNA polymerase.
IGBMC-CERBM Strasbourg provides facilities for protein expression in Yeast. Yeast cells constitute a convenient alternative host for production of either recombinant eukaryotic proteins or endogenous complexes. Advantages include the low cost of growing material, robustness of the system, easy genetic manipulation, and the presence of endogenous eukaryotic-type protein modification and chaperone systems
The high throughput crystallization platform offers vapour diffusion as well as crystallization under oil screening and optimization in sitting drops. Flexible methods have been developed to adapt each step to individual project requirements. At the crystallization level, we have especially focused on developing techniques to optimize crystal growth.
Ultra high-intensity microfocus rotating anode X-ray generator coupled with a diffractometer for collection of diffraction data from single crystals, and a BioSAXS device for collection of diffusion data from macromolecular solutions. The variable divergence on the optics, coupled with a pixel array detector, allows for the resolution of unit cell dimensions up to 600Å.
An Isothermal Titration Calorimeter (ITC) is used to quantify the binding of ligands relevant in the case of work with e.g. receptors and transporter.
Measure changes of mobility of the molecules in microscopic temperature gradients to determine binding affinities
Multi-angle and Dynamic Light Scattering atIGBMC-CERBM
A Rigaku BioSAXS-1000 is attached to the right port of a Rigaku MicroMaxTM-007HF generator. The BioSAXS-1000 is fitted with Osmic optics and a 2D Kratky block for collimation. Data is collected on a Pilatus 100K photon counting detector (Dectris).
The Leica SR GSD microscope allows a range of localization super-resolution experiments (dSTORM, GSDIM, STORM, PALM), particle tracking, standard wide-field epifluorescence and TIRF microscopy, transmission microscopy with polarization contrast. Samples can be mounted either on standard slides or in 35mm diameter glass bottom petri dishes.
Thermal shift assay is a thermodenaturation assay to monitor the thermal stability of proteins and investigate factors affecting this stability. This rapid and simple technique is used in high-throughput mode to screen optimal buffer conditions, ligands, cofactors and drugs for purified proteins.
The Strasbourg center provides a cutting-edge technological environment for integrative structural biology approaches, from the molecular to the cellular level, with an emphasis on protein production linked with preparation of samples for EM and crystallography.
Protein engineering and production are essential tools for structural, biophysical and functional studies as well as for biotechnology and medical applications. The Strasbourg center offers a state-of-the-art infrastructure for the production of biomolecules and biomolecular complexes in E. coli, yeast, insect cells (baculovirus expression system) as well as in mammalian cells using an attenuated vaccinia virus vector. The Strasbourg platform has a unique expertise on production of recombinant multi-protein complexes as well as on purification of endogenous complexes using the TAP-tag technology in yeast.
Strasbourg provides core-technologies for structure determination by crystallography, cryo-EM and tomography. Strasbourg is a center of expertise for crystrallographic analysis of protein-protein and protein-nucleic acid complexes (nano and micro-automated screening and HTP crystal production, data collection and structure determination) and cryo-electron microscopy/tomography (Hight resolution cryo-EM, cryo electron tomography using up to date equipment (Polara and Titan microscopes and Falcon detectors)
The center also offers access to
- biophysical plateform to monitor protein properties and function, including protein stability, protein complex formation, oligomerisation and protein interactions,
- shape determination by SAXS.
- NMR (700MHz, 600MHz with cryo-probe, protein and nucleic acid isotope labelling, solution structure determination & dynamics, translational diffusion measurement)
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