High-throughput crystallisation lab
Figure 1: Detail of the PYR1 Abscisic acid receptor dimerisation interface
Figure 2: Structure of the Abscisic acid hormone receptor showing the gating loops in the closed (magenta) and open (green) conformations.
The Márquez team develops low volume, high-throughput, techniques to optimise protein crystallisation and uses them to study the structure of sensing and signalling molecules.
Previous and current research
The HTX lab is one of the major facilities for high-throughput nanovolume crystallisation screening in Europe, open to scientists working in European academic institutions through the EC-funded Protein Production Platform project P-CUBE. It is also one of the major resources of the Partnership for Structural Biology in Grenoble. Since opening, the HTX lab has offered services to hundreds of scientists, performing several million experiments. The lab is also involved in the development of data management resources, as well as new crystallisation techniques.
Towards the integration of automated crystallisation and data collection: While crystallisation and X-ray data collection is often highly automated, the process of mounting crystals into cryogenic X-ray data collection supports remains difficult and time-consuming. In collaboration with the Cipriani team, we have developed a new approach (known as Crystal Direct) designed to enable full automation of crystal harvesting process, based on a redesigned vapour diffusion crystallisation plate. Advantages include: direct compatability with X-ray data collection; recovery using laser-induced photo ablation; absence of mechanical stress during crystal mounting; and the possibility to recover micro-crystals. A prototype has been built and is currently being tested.
Crystallisation Information Management System (CRIMS): CRIMS tracks experiments and makes results available to users via the web in real-time. We developed the system to tackle the challenge of capturing the enormous amounts of information generated by modern experiments. CRIMS software is currently in use at six other laboratories in Europe, three of them at synchrotron sites.
Molecular mechanisms in sensing and signalling: Our research focus is on understanding the mechanisms of sensing and signalling at a structural level. Recently, we have obtained the structure of the receptor for abscisic acid (ABA), a hormone regulating the response to environmental stress in plants. We have shown how receptor dimerisation modulates ligand binding affinity leading to differential sensitivities towards the hormone (Dupeux et al., 2011). The work provides a novel framework for understanding the ABA signalling pathway and activation of the stress response in plants, and illustrates how receptor oligomerisation can modulate ligand binding affinity by influencing the thermodynamics of the overall activation reaction.
Future projects and goals
Aside from the continuous development of our crystallisation plates and crystal harvesting system, we will focus on the implementation of automated cryo-protection protocols as well as in the connection of CRIMS with synchrotron data management systems, with the aim to fully integrate crystallisation and data collection.
We continue to focus on the structural study of signalling systems. The structure of the ABA hormone-receptor complex paves the way for the design of small molecules able to bind to the ABA receptors and activate the stress signalling pathway. Such molecules should be easier to synthesise and more stable than ABA itself and could be used to improve crop tolerance to drought and other types of environmental stress.