Mostly motivated by, but not restricted to the comprehensive analysis and understanding of biological phenomena with potential medical implications, structural biology gives access to the atomic details of macromolecules, allowing to decipher their biological functions. Using state-of-the-art developments coupled to third-generation x-ray synchrotron sources, macromolecular x-ray crystallography (MX) stands as the primary method for determining the structures of proteins, alone or in complex with partner ligands. The major bottleneck of MX lies in the requirement to obtain crystals of reasonable sizes that can easily by handled and readily give rise to interpretable diffraction patterns. Prior to x-ray diffraction studies, crystal production pipelines involve the characterisation, purification and handling of samples in large quantities through multi-step, complicated, time-consuming and costly procedures. The identification of protein crystals naturally occurring inside cells and organisms as diverse as bacteria, protists, fungi, plants, fishes, amphibians, insects and mammals has opened a window for a new type of MX and structural biology. Recently, the emergence of the in vivo crystallography (ivMX) approach took advantage in the developments of new intense coherent x-ray sources that allow collecting diffraction patterns from sub-micron crystals, targets so far unreachable at other x-ray sources. This proposal intends to get further insights into the yet uncontrollable events dictating in vivo crystal growth, by structure determination and analysis of readily available ivMX systems. While deciphering these phenomena and applying them to external proteins recombinantly expressed in hosts where in vivo crystal growth could be identified, a small platform for ivMX will be initiated, with the aim of reducing the tedious and costly sample preparation steps currently used in MX.

Correlative multimode imaging is the only way to reveal a composite view of a biological sample with the multidimensional information about its macro-, meso- and microscopic structure, dynamics, function and chemical composition that is required in order to understand biomedical processes and diseases. Project CLEXM addresses an urgent need to demonstrate, promote and disseminate the benefits of this technique in the fields of disease and drug therapy research and especially to early-career researchers. The premise of project CLEXM is that there is a growing need for disease and drug therapy researchers to understand the linkages between structural and functional changes that occur in a cell and to be able to observe these from the cellular (micrometre) to the molecular (nanometre) scale. Correlative Light and Electron Microscopy (CLEM) is the current state-of-the-art for achieving this, but the technique is extremely complex and slow. CLEXM postulates that the integration of a third imaging modality, Soft X-ray Tomography (SXT), into CLEM will make it easier and faster for researchers to correlate cellular structure with cellular function. Correlative Light, Electron and X-ray Microscopy (CLEXM) can be combined in a number of ways and the benefits will be demonstrated in a number of different use cases. This would be too difficult and too much to achieve as a single research project or as a single MSCA Postdoctoral Fellowship, however, it lends itself to be most easily achieved as a network of complementary individual projects, under an MSCA Doctoral Network action. The overarching objective of project CLEXM is to provide high-level training in the field of correlative multimode imaging to a new generation of doctoral candidates to provide them with the transferrable skills necessary for thriving careers in a high-growth area that will aid researchers in their quest to understand disease and to develop effective therapies.