Biology

Dr. Sandip Kaledhonkar
Columbia University, New York, USA

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 and recycling process 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.

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.