Márquez Team
The high throughput crystallisation lab at the PSB
High-throughput crystallisation robot at the HTX Lab.
Structure of the Abscisic acid hormone receptor showing the gating loops in the closed (magenta) and open (green) conformations.
Previous and current research
Automated crystallisation: Obtaining crystals from biological molecules is one of the major bottlenecks in structural biology. Once macromolecules are purified, they need to be assayed for crystallisation with a collection of precipitants under different chemical environments, which can consume large amounts of sample and time.We have established a fully automated high-throughput crystallisation screening facility, the HTX lab, which processes large numbers of samples using very low sample volumes. Since its opening, we have offered services to hundreds of scientists performing several millions of experiments. The HTX lab is next to the ESRF synchrotron and offers crystallisation screening services to scientists working in European research institutions through the EU-funded FP7 programme PCUBE.
One of the major problems we face is accurately capturing the enormous flux of information generated by experimental parameters and results. For this purpose , we have developed the Crystallisation Information Management System (CRIMS), which tracks experiments and makes results available to users via the web in real-time along with all the experimental parameters. It is also synchronised with the automated data collection systems at the ESRF and has been distributed to other European labs. The HTX lab not only increases efficiency in the process of structure solution, but also represents a technological advantage critical for the success of challenging projects like those studying protein complexes and large macromolecular assemblies.
Molecular mechanisms in Sensing and Signaling: Our research focus is in understanding the mechanisms of signaling at a structural level and specially sensing processes. In the past we have contributed to the understanding of the mechanisms of action of theHPrKinase / phosphatase, a sensor of the metabolic state in bacterial cells. More recently, we have obtained the structure of the Abscisic Acid (ABA) receptor, a hormone regulating the response to environmental stress in plants. This receptor belongs to the so called PYR/PYL/RCAR, family and is able to bind ABA and inhibit the activity of specific protein phosphatases of the type 2C (PP2Cs) leading to the activation of the signaling pathway controlling the stress response. Our work shows how the hormone is bound in a large cavity inside the PYR1 ABA receptor. The loops surrounding the entry to this cavity act as ‘gates’, closing over the hormone once it is inside. Amino acids in these same loops are also important for the interaction between PYR1 and the phosphatases, implying that binding of the hormone to the receptor and the closure of the gating loops generates an optimal surface for binding of the PP2Cs. This work represents the definitive confirmation of the PYR/PYL/RCAR protein family as ABA receptors, but also provides insights into the basic mechanism of hormone sensing.
Future projects and goals
Developing new technologies in crystallization: With the new EC-funded PCUBE programme giving access to the platform to European scientists we will extend the base of HTX lab users .We will also develop an improved version of the CRIMS data management software specially designed to allow easy installation and customization in other labs. In collaboration with the Synchrotron Instrumentation Group in Grenoble and based on solutions we have already explored and patented, we plan to develop an integrated screening station where users can bring either the protein in solution or crystallisation trays, and get X-ray data with minimum input. This will require the development of new crystallisation supports and crystal harvesting robotics.
In the laboratory, we will continue the study of signaling systems with a special focus on two major fields: TEC-family protein tyrosine kinases and the ABA signaling pathway. 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 signaling pathway. These molecules should be easier to synthesize and more stable than ABA itself and could potentially be used to improve the tolerance of crops to drought and other type of environmental stress. We will also focus on the structural study of other components of the ABA signalling pathway.