The functional and mechanistic understanding of biological macromolecular complexes requires methods to investigate their molecular composition, arrangement, and conformational landscape in a close-to-native condition. Cryo-electron microscopy (cryo-EM) is now a powerful tool for studying the structure of biological specimens by imaging the macromolecules of interest vitrified within biochemically functional buffers. However, many macromolecules prove to be difficult to prepare in the form of cryo-EM specimens and further improvement to obtain cryo-grids of sufficient quality for high-resolution analysis often requires the careful optimisation of a wide range of variables. Therefore, the current challenges and limitations of this method are linked both to the intrinsic nature of the macromolecular complexes that we want to analyse and to the currently available instruments and workflows.
Our team will develop high-throughput and fully automated pipelines to enable large-scale cryo-EM sample preparation and screening. This multidisciplinary project will integrate the work of engineers, software developers, image analysists, and molecular biologists developing new approaches to address some of the major rate-limiting steps that currently affect the throughput and the success rate of cryo-EM projects. Our pipeline will integrate specimen handling, sparse-matrix screening, and biochemical characterisation workflows to explore sample stability within a wide range of buffer conditions. The produced screening conditions will be applied to a range of cryo-EM supports by newly developed vitrification devices able to provide controlled and efficient preparation of cryo-grids. We will integrate the existing imaging routines with new software applications to establish a fully automated pipeline that will cover all the imaging steps required for large-scale cryo-EM sample screening. Our pipeline will rely on minimal operator dependency and will provide full tracking of the samples throughout the entire process. We will establish a dedicated data management system implemented with a secure web interface for project planning and real-time remote inspection of the results.
The Mattei team is part of the EMBL Imaging Centre, which represents EMBL’s new service unit for cutting-edge electron (EM) and light microscopy (LM) technologies, including academically developed methods not yet commercially available. The main mission of the EMBL Imaging Centre is to make the most advanced microscopy technologies available to the broad scientific international user community from both academia and industry as quickly as possible. A powerful and synergistic portfolio of imaging technologies will be offered both individually and in a combined (correlative) fashion, to enable new ground-breaking research that crosses the scales of biology.