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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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INSTRUCT CENTER - FRANCE 1 - IGBMC

HIGH THROUGHPUT CRYSTALLISATION

The high throughput crystallisation 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 crystallisation level, we have especially focused on developing techniques to optimize crystal growth.

Crystallisation is performed using general screens from commercial sources as well as specific screens made from our own stock solutions.

Sample characterization is a prerequisite before crystallisation. Unless it has already been performed, samples will be analyzed by native and SDS-PAGE, as well as dynamic light scattering and thermal shift assay. The platform also offers analysis by analytical ultracentrifugation.

Hanging drop plates and capillaries for free interface diffusion are also proposed for manual screening and optimization.

The process is automated at different steps:

- Home made and optimization screens are prepared with a Tecan Evo Workstation,

- Sitting drops -from 100nl to 500nl- are performed by a Mosquito robot,

- Batch crystallization in microfluidics chip are set up using a Plugmaker Microcapillary Protein Crystallization System™,

- Plates are stored and automatically imaged at 4°C and 20°C in Rock Imager systems (Formulatrix). Images are accessible to users and can be scored via a web-interface. Plates can also be stored at 4°, 20° and 26°C and inspected through a Leica stereo-microscope.

Devices:

A Tecan Evo 75 pipetting station is used to prepare optimization screens in 96-well deep-well blocks.

A Liquidator 96 Manual Pipetting System is used to dispense the reservoirs.

Crystallization droplets are set up by a Mosquito Crystal robot.

A PlugMaker Protein Crystallization Workstation is used to perform crystallization in microfluidics chips.

For more details see: https://www.structuralbiology.eu/update/platform/high-throughput-crystallisation-at-igbmc-cerbm-in-strasbourg

BIBLIOGRAPHY

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Kolaj-Robin OMcEwen AGCavarelli JSéraphin B. Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification. FEBS J. 2015 Mar;282(5):819-33. doi: 10.1111/febs.13199. Epub 2015 Feb 4.

Rousseau A, McEwen AG, Poussin-Courmontagne P, Rognan D, Nomine Y, et al. (2013) TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration. PLoS Biol 11(12): e1001726. doi:10.1371/journal.pbio.1001726.

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Susini L, Besse S, Duflaut D, Lespagnol A, Beekman C, Fiucci G, Atkinson AR, Busso D, Poussin P, Marine JC, Martinou JC, Cavarelli J, Moras D, Amson R, Telerman A. (2008) TCTP protects from apoptotic cell death by antagonizing bax function. Cell Death Differ. 15:1211-20.