Figure 1: The CrystalDirect technology for automated crystal mounting and cryocooling

Figure 1: The CrystalDirect technology for automated crystal mounting and cryocooling.

Figure 2: Fragment based screening enables the identification of active site and allosteric site molecules binding Trypanosoma´s FPPS protein, opening new possibilities for the development of antiparasitic therapies

Figure 2: Fragment based screening enables the identification of active site and allosteric site molecules binding Trypanosoma´s FPPS protein, opening new possibilities for the development of antiparasitic therapies (in collaboration with Wolfgang Jahnke, Novartis, Chembiochem. 2020 Jun 14).

The Marquez Team develops methods and software for high-throughput, fully automated crystallography and fragment screening, using them to study biomedically relevant systems.

Previous and current research

The Marquez Team operates the High-Throughput Crystallization Laboratory (HTX Lab), one of the major facilities for high-throughput, nanovolume, crystallization screening in Europe and one of the major resources of Grenoble’s Partnership for Structural Biology. His Team has a strong focus in the development of new technologies in macromolecular crystallography, including methods for sample evaluation and quality control and the CrystalDirect technology (see figure), enabling fully automated crystal mounting and processing. This technology is based on the use of a modified vapour diffusion crystallization microplate and a laser beam to mount and cryo-cool crystal samples into diffraction compatible supports closing the automation gap existing between crystallization and data collection.  The HTX lab has also developed the Crystallization Information Management System (CRIMS), a web-based laboratory information system that provides automated communication between crystallization and synchrotron data collection facilities, enabling uninterrupted information flow over the whole sample cycle from pure protein to diffraction data.

Online crystallography and fragment screening pipelines

Through the combination of the CrystalDirect technology and the CRIMS software the HTX lab has developed the concept of Online Crystallography: Fully automated, remote controlled crystallography pipelines integrating crystallization screening, crystal optimization, crystal mounting and cryo-cooling and automated X-ray data collection (at ESRF and PETRA III synchrotrons) into continuous workflows controlled though dedicated web interfaces. This approach minimizes the delay between crystal growth and measurement, accelerating the progression of very challenging projects that require diffraction-based optimisation of protein production and crystallization conditions and frees scientists from complex operations associated with crystallization, crystal handling and beamline operation, rendering crystallography more accessible to non-expert groups.

The CrystalDirect technology enables automated delivery of small molecules to crystal. Based on this approach we have developed an automated fragment screening and analysis pipeline enabling the analysis of libraries with over 1000+ fragments. These pipelines can also be applied to the rapid structural characterization of protein-ligand complexes. X-ray diffraction data collection is typically carried out at the MX beamlines of the European Synchrotron Radiation Facility (ESRF) operated by the  ESRF- EMBL Joint Structural Biology Group. We apply these pipelines to the study of biomedically relevant proteins (see figure)

Future projects and goals

Our focus is in the development of innovative approaches for crystallographic compound and fragment screening, that we apply to the study of biomedically relevant proteins. In collaboration with the Synchrotron Diffraction, Instrumentation and ESRF MX Teams we are working towards integrating the CrystalDirect technology into the MASSIF-1 beam line environment, which will now be operated in coordination with the HTX Lab. This will increase our throughput and capabilities to support fragment screening and online crystallography. At the same time, it will enable new experimental opportunities. Moreover, we will continue to develop the CrystalDirect technology to support serial crystallography applications as well as new approaches for the rapid analysis of membrane proteins both at cryogenic and room temperature. On the software side we continue with the development of the CRIMS software, particularly in the area of large-scale fragment data analysis. For this we benefit from a long-standing collaboration with the Global Phasing Ltd. Team as well as from an extended network of collaborators in academia and industry.

Our interdisciplinary team offers opportunities for scientists, engineers and software developers to work in one of the leading infrastructures for structural biology within the areas of protein crystallography, drug design, automation, and large-scale scientific data management and analysis. Currently, we are particularly interested in profiles in structural biology or computer science orientated towards one or several of the following areas: Protein Crystallography, fragment screening, structure guided drug design, cloud computing, machine learning and artificial intelligence.