Figure 1: The CrystalDirect technique could benefit challenging structural biology projects, such as studies of membrane proteins or multi-protein complexes.
Figure 2: Detail of the Fra a 1 and Fra a 3 proteins in apo and ligand bound forms respectively.
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 High-Throughput Crystalisation (HTX) Laboratory 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. It offers services to scientists working in European academic institutions through the EC-funded iNEXT and BioStructX projects and, at the same time, develops novel approaches in macromolecular crystallisation.
Integration of crystallisation and synchrotron data collection facilities through automated crystal harvesting and processing
In collaboration with the Cipriani Team, we developed CrystalDirect™, a fully-automated method for harvesting and processing crystals (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; reduced mechanical stress during mounting; and compatibility with X-ray data collection. CrystalDirect was recently upgraded with an automated sample cry cooling and ligand soaking. The first prototype is now in operation at the HTX lab and is available to users through remote web interfaces (Marquez & Cipriani, 2014).
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. The analysis of the data stored in 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). We constantly implement new functionalities to improve users’ capabilities (see figure).
Molecular mechanisms in sensing and signalling
Our research focuses on the mechanisms of sensing and signalling at a structural level. We have recently contributed to the study of the function of plant members of the START family, including the PYR/PYL abscisic acid (ABA) receptors involved in the response to abiotic stress and the PR-10/Fra a proteins, which are involved in the control of secondary metabolic pathways during fruit ripening.
Future projects and goals
In collaboration with other EMBL and ESRF groups we will work to develop a new generation of highly automated pipelines. These will integrate crystallisation, crystal harvesting and processing, and data collection in a single, automated workflow. This will enable us to implement fully automated pipelines for large scale ligand and fragment screening through macromolecular crystallography and to provide fully automated, remotely operated crystallography services to support the structural analysis of very challenging targets. Funded access to these new pipelines will be available through the EC-funded iNEXT project.