Title: Structural Dynamics of Biomolecules Captured By Time-resolved Cryo-Electron Microscopy

Speaker: Dr. Sandip Kaledhonkar, Columbia University

Abstract: Structural dynamics of biological macromolecular is crucial to its structure-function relation, hence obtaining high resolution structures of the biomolecules is an essential task. The structure determination of biomolecules was dominated by x-ray crystallography over last few decades. In contrast, single-particle cryo-electron microscopy (cryo-EM) was mainly employed for understanding morphology of the biological macromolecules until 2012. With the advent of direct electron detector camera, cryo-EM has become a popular choice for biomolecular structure determination as it can, (i) obtain near atomic resolution of biomolecules and, (ii) determine multiple structures or conformations co-existing in one sample. The grid preparation for cryo-EM takes several seconds. However, some biological processes are much faster than second time scale, and the ensuing short-lived states of the molecules are difficult to capture. To address this issue, we developed time-resolved (TR) cryo-EM using microfluidic chip to trap short lived intermediates from tens of milliseconds to second time window. In this presentation, I will discuss TR cryo-EM technique along with single particle analysis to visualize transient intermediate structures of translation initiation during protein synthesis. While translation initiation phase of protein synthesis, a small, or 30S, subunit and a large, or 50S, subunit, join together to form a 70S initiation complex (IC) ribosome with the help of initiation factors. We have resolved discrete structural intermediates on the 50S subunit joining and 70S IC formation pathways with a time resolution of tens of milliseconds. Our study reveals the time scale of dissociation of initiation factors and its implication towards translation initiation process. The work presented here demonstrates the powerful ability of time-resolved cryo-EM to reveal the structures of transient intermediates on the reaction pathways of biomolecular machines as they execute some of the most fundamental processes in all of biology.