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.
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 CrystalDirect technique could benefit challenging structural biology projects, such as studies of membrane proteins or multi-protein complexes
Previous and current research
The HTX lab is one of the major facilities for high-throughput nanovolume crystallisation screening in Europe and one of the major resources of Grenoble’s Partnership for Structural Biology, open to scientists working in European academic institutions through the EC-funded P-CUBE and BioStructX projects. The lab has offered services to hundreds of scientists, performing several million experiments. We are involved in the development of data management resources and new crystallisation techniques.
The Crystallisation Information Management System (CRIMS): CRIMS tracks experiments and makes results available to users via the web in real-time, along with all experimental parameters. It has been licensed to 10 other laboratories in Europe, three of them at synchrotron sites. Recently, data mining through CRIMS has allowed us to develop a new method to determine the crystallisation likelihood of a protein sample based on a simple assay measuring thermal stability (Dupeux et al., 2011).
Integration of crystallisation and synchrotron data collection facilities: While both highly automated platforms, recovering crystals and mounting them on supports remains a difficult and time-consuming manual process. In collaboration with the Cipriani Team, we developed Crystal Direct™, an approach that enables full automation of the crystal harvesting process (see figure). Crystals are grown on an ultrathin film in a vapour-diffusion crystallisation plate and recovered through laser-induced photo ablation. Advantages include: elimination of crystal fishing and handling; absence of mechanical stress during mounting; and compatibility with X-ray data collection. The first prototype is now in operation and could benefit many projects.
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 and shown how receptor dimerisation modulates ligand binding affinity leading to differential sensitivities towards the hormone (Dupeux et al., 2011). This provides a 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
In collaboration with the McCarthy and Cipriani teams we will link the crystallisation screening service with the automated evaluation of crystals in situ with X-rays. New interfaces will be added to CRIMS to allow remote operation and we aim (with collaborators) to integrate CRIMS with synchrotron data management systems. We will develop vapour diffusion and microfluidic devices for crystal optimisation experiments. We aim to establish CrystalDirect for routine use in macromolecular crystallography and a series of pilot projects will be selected to establish standard protocols and develop new applications. Prototypes of the new plates will be distributed to other labs as part of the INSTRUCT project, and we will work towards the development of promising new approaches for crystal processing, including for cryo-protection, crystal soaking and crystal freezing. Towards this goal, an advanced version of the CrystalDirect harvester will be designed in collaboration with the Cipriani team.