Development of Serial electron and X-ray nano-crystallography by use of in vivo grown nanocrystals. – SNaX

Serial electron diffraction (SED) and serial synchrotron nano-crystallography (nano-SSX) are just-emerging methods in structural biology, whereby data are collected from protein nanocrystals using a sub-micron-sized electron or X-ray beam, respectively. These methods are set to lead the future of nano-crystallography, given their cost-effectiveness and the small amount of sample required. Combining the expertise of the Colletier, Lereclus and Miller teams, the project will address four issues of fundamental importance to further advance the SED and nano-SSX methodologies. Specifically, we will (i) test if the methods can be applied to solve nano-crystalline protein structures in the cellular environment (i.e. in vivo); (ii) determine what is the smallest crystal size that can be probed by SED and nano-SSX; (iii) develop an in vivo crystallization approach based on recombinant expression of fusion proteins in the crystalliferous bacterium Bacillus thuringiensis; and (iv) offer a proof of feasibility for time-resolved SED experiments on macromolecular nanocrystals. Results from the SNaX project will allow to explore the boundaries of SED and nano-SSX, and to develop their full promises for nano-crystallography.