Frontier Research Institute for Interdisciplinary Sciences
Tohoku University

Cryogenic electron microscopy (cryo-EM) single-particle structural analyses of protein and RNA complexes have evolved dramatically due to technological advances in direct ion detector as well as computational analysis methods. The final resolution of the map is now reaching to sub-angstrom resolution, exceeding 2Å, however, it is only the case of stable protein complexes with little fluctuation. For instance, enzymes, which control the function of proteins and molecules, bind to their targets and rapidly release after reaction, making it difficult to capture reaction complexes.

In this research project, we will focus on sample preparation methods for cryo-EM structural analysis, and aim to develop stabilization techniques for reaction complexes using chemical modification of protein residues and cross-linking reagents, and to apply them to structure visualization techniques combined with cryo-EM single particle structural analysis. (1) Ikeuchi will prepare samples for structural analysis by using genetics and biochemistry, adding genomic tags, purifying complexes, and constructing an in vitro reaction reconstruction system. (2) Sato will optimize and apply his originally developed high-reactive chemical modifier to protein crosslinking for cryo-EM samples. (3) Visualize the structure of intracellular complexes with large fluctuations by cryo-EM single-particle structure analysis of cross-linked samples (Ikeuchi). (4) Analysis and evaluation of the extent and efficiency of modification (cross-linking) introduction will also be performed by mass spectrometry (Sato).

This technology is expected not only to enable structural analysis of high reaction rate complexes by capturing the movement of various proteins and RNAs, but also to directly contribute to life science, drug discovery development, and fundamental research on disease pathogenesis mechanisms.