Crystallization of membrane proteins with an automated microfluidic pipeline (ESR2)

Applications are invited for an Early Stage Researcher (ESR)/PhD studentship positions funded by The Marie Skłodowska-Curie Actions (MSCA) Innovative Training Networks (ITN) programme “Rationalising Membrane Protein Crystallisation” (RAMP) and based in the Synchrotron Group at the Institut de Biologie Structurale (IBS), France. The Institut de Biologie Structurale (IBS) is a research centre for integrated structural biology funded by the CEA, the CNRS and the Université Grenoble Alpes (UGA).

Over the last 20 years, and while the crystallisation of membrane proteins remained almost entirely empirical, a combination of experimental and computational work greatly advanced the understanding of the nucleation and growth of crystals in many other systems, from ice to globular proteins. Here we aim to build on this work to apply predictive models and microfluidic techniques to membrane protein crystallisation to tailor crystal sizes and morphologies required for different downstream structure determination approaches. This PhD project is one of 12 in the RAMP training network. Other PhD students in the RAMP network will develop advanced methods for crystallisation and modelling of the membrane protein crystallisation process and apply it to the structure determination of important membrane proteins.

The idea is to move away just from conventional miniaturisation of the crystallisation experiment via smaller drops, and to focus on miniaturising apparatus that allows precise control of the experiment, unlike vapour diffusion. We have already produced a device that allows batch temperature controlled crystallisation to grow large crystals for neutron diffraction experiments (Budayova-Spano et al., 2007 Acta Crystallogr. D, 63(3), 339-347). The goal now is the precise and reversible control of crystallisation via dialysis and temperature, but at the nano scale. This will have important benefits, especially for membrane proteins where both protein and detergent are expensive. It will also be extremely valuable for in meso or HILIDE crystallisation, as it would require a thorough exploration of the crystallisation phase space to find the optimal way of getting crystals.

We have already developed a crystallisation bench that provides temperature and dialysis control and uses just 15 μl (Junius et al., 2016, J. Applied Cryst., 49, 806-813); the technological goal of this PhD project is to pursue miniaturisation of this technology into microfluidic chips with the volume of protein sample down to 200 nL, and allow direct visualisation. Thus, the PhD project focuses on the development of an integrated crystal growth optimisation platform that incorporates a microfluidic temperature-controlled optimisation lab-on-a-chip equipped with a microscope-mounted video camera allowing for a real-time observation as well as on an easy-to-use software interface that allows the user to visualise the crystals, process images and control the temperature and mother liquor composition from one interface. The major challenge is the integration of fluid handling capabilities enabling rapid mixing for reaction initiation. The chip should of course be X-ray compatible, allowing in situ X-ray diffraction. Finally, the aim is to use the developed microfludidc pipeline to grow a large number of protein crystals of model and biologicaly challenging proteins for serial X-ray crystallography and time-resolved crystallography.

The project will involve secondments to other network partners with specialised membrane protein crystallisation, microfluidic and crystallography expertise such as Trinity College Dublin, University of Aarhus, AstraZeneca, CNRS Bordeaux and the Hamburg Centre for Ultrafast Imgaging, and cosupervision of Molecular Dimensions Ltd.

This project will involve crystallisation and in situ X-ray diffraction analysis of crystals of model as well as difficult membrane protein targets, as well as instrumentation, automation and methodological development in microfluidics and protein crystallography, with support from the existing expertise in the group. Crystals will be studied at national and international synchrotrons, neutron sources and free electron lasers.

We are looking for an enthusiastic academically qualified (bio)physicist, physico-chemist or engineer with a strong interest in structural biology and multi-disciplinary problems. Experience in macromolecular crystallization and or crystallography is an advantage. Given the scope of the project and the need for a close collaboration with other Consortium members as well as with the industrial/external partners excellent communication skills and the ability to work as part of a team are prerequisites.

Applicants must have the background and expertise required for the position, be in the first four years full-time equivalent research experience of research career, since, e.g., completion of their masters’ degree, do not already possess a doctorate degree, be willing to move to FRANCE and to other countries within the network, they must also be eligible for a working permit for FRANCE in which they have not lived for more than 12 months over the last 3 years (short stays, such as holidays, are not taken into account), be proficient in both written and spoken English.

Candidates must comply with EU and Université Grenoble Alpes eligibility criteria. Due to the EU rules to promote mobility, you are not eligible for a position in a country where you have lived (worked, studied) for more than 12 months in the last 3 years. So for this position you are eligible, unless you have studied or worked in France for more than 12 of the last 36 months. For applicants finishing or who have just finished their degree, this typically means that you can be graduating from any university except a French university.

Further details on this project are available from Dr. Monika Spano (monika.spano@ibs.fr).

Author: Richard Sear

Computational physicist at the University of Surrey. My research interests are in crystallisation, soft matter & biological physics