Uncovering hidden structures in protein crystallization with coherent X-rays

Diffraction and serial crystallography serve as essential tools for comprehending the functions and dynamics of proteins. However, a significant limitation to their application lies in the consistent generation of protein crystals. Thus, it becomes imperative to explore novel avenues that can facilitate and regulate protein crystallization. Classical nucleation theory does not encompass the crystallization pathway for numerous protein solutions. This proposal seeks to offer fresh experimental insights into hidden intermediate metastable structures that are hypothesized to occur during the observed anomalous crystallization mechanism. Our objective is to explore in a non-equilibrium manner the anomalous crystallization of ferritin solutions in microdroplets and characterize its time evolution. The latter is going to be accessed via X-ray Photon Correlation Spectroscopy (XPCS), which will reveal the dynamic properties of both proteins and the amorphous phase that precedes crystal formation. Additionally, we aim to quantitatively assess the role of water in this system, pertaining to protein-protein interactions, hypothesized to depend on the water structuring in the vicinity of the interacting surface. Capturing the desolvation process is crucial for the understanding of the molecular mechanism underlying the different crystallization pathways, since it has been hypothesized that the efficiency and directionality of the desolvation leads to a higher (‘classical’) or lower (‘nonclassical’) degree of protein ordering in the initial aggregates. The candidate has a strong background in experimental methods sensitive to protein dynamics and joins the host group which specializes in water experiments with microdroplets. The project utilizes the unique coherent properties of European large scale facilities, such as the European Synchrotron Radiation Facility (ESRF) and European X-ray Free Electron Laser (Eu-XFEL).