POSTPONED - Finding the best methods and conditions for growing large enough crystals that diffract well is a challenge in protein crystallography - Postponed

Start Date
25-03-2020 10:00
End Date
25-03-2020 11:00
Room 337, Central Building
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Eleanor Ryan
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Finding the best methods and conditions for growing large enough crystals that diffract well is a challenge in protein crystallography. Protein crystals are used in different diffraction techniques and therefore we need crystals of different sizes. For neutron protein crystallography, crystals with a volume larger than 0.1mm3 are required. However, for serial X-ray crystallography crystals of a few micrometers in size are sufficient [1]. Different physico-chemical parameters can affect crystallization. Different crystallization methods can also play a crucial role in protein crystallization, as they use different kinetic pathways to reach nucleation and metastable zones in the crystallization phase diagram. To obtain suitable crystals with the required sizes for different crystallography methods, a crystallization bench was developed in our lab [2]. It already allowed us to obtain crystals of good quality for neutron and X-ray diffraction [1,2]. The crystallization bench enables us to control and vary the temperature as well as the chemical composition of crystallization solutions (e.g. concentration of crystallization agents) through dialysis during the experiment. The goal of my work is to develop the current crystallization bench as well as the rational optimization strategies of crystal growth based on knowledge of equilibrium phase diagrams for the crystallization of membrane proteins.

To apply our approach to the membrane proteins, we designed crystallization experiments to compare different crystallization techniques in order to optimize the crystallization of biological macromolecules. For this study, two model membrane proteins are used. ShuA [3] from Shigella dysenteriae (22-stranded transmembrane β-barrel transferring heme across the outer membrane) and AcrB [4] multidrug efflux pump transporter in Escherichia coli (a homotrimer protein located in the inner membrane containing 12 transmembrane helices and 2 large periplasmic domains).

Both proteins were successfully purified and crystallized with different crystallization techniques including vapor diffusion, dialysis, and batch methods. The sets of diffraction data for AcrB crystals were collected on the beamline BL13 – XALOC at the ALBA.

In the series of experiments that we performed, crystals grown with dialysis are uniform and demonstrate almost the same diffraction quality, in contrast to vapor diffusion, where significant diversity of crystals and diffraction qualities coexist within a single sample. We aim to collect much more datasets for a statistical comparison between these methods and we expect to crystallize and improve the crystal quality of both proteins using our crystallization bench as well as to collect preliminary diffraction data using neutron diffraction.

In addition, systematic dynamic light scattering measurements will be performed to characterize our protein solutions prior to the crystallization process. From these studies, we will obtain information on the hydrodynamic radius, the diffusion coefficient and the polydispersity, important parameters that will tell us about the purity and the homogeneity of the proteins in solution used for further crystallization experiments.

[1] Junius, N., Vahdatahar, E., Oksanen, E., Ferrer, J.L., & Budayova-Spano, M. Optimization of Crystallization of Biological Macromolecules using Dialysis Combined with Temperature Control (accepted for publication in J. Appl. Cryst.)

[2] Junius, N., Oksanen, E., Terrien, M., Berzin, C., Ferrer J.L., & Budayova-Spano, M. A crystallization apparatus for temperature-controlled flow-cell dialysis with real-time visualization. J.Appl. Cryst. (2016), 49(3), pp.806-813.

[3] Brillet, K., Meksem, A., Thompson, A. and Cobessi, D., Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae. Acta Cryst. (2009). F65, 402–405.

[4] Murakami, S., Nakashima, R., Yamashita, E. and Yamaguchi, A., Crystal structure of bacterial multidrug efflux transporter AcrB. Nature, (2002). 419(6907), pp.587-593.

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