Biology

Dr. Sandip Kaledhonkar
The Frank Lab Columbia University Medical Center, New York

Abstract:

Following recent advances of recording media, single-particle cryo-EM has become a popular choice for structural biologists studying biological macromolecules. The high-resolution Coulomb potential maps obtained by 3D reconstruction allow biomolecular interactions to be interpreted on near-atomic levels. Most of the new structural data have been obtained from molecules in post-equilibrated states, without an attempt, or a means, to follow a reaction in real time. Time-resolved (TR) cryo-EM provides a way to study the progress of a reaction between two components that goes through one or several intermediate short-lived states before arriving at the final product. We adopted and further developed an experimental setup in which two reactants are allowed to mix and react inside a microfluidic chip, then sprayed onto the grid [1].  In my talk, I will briefly discuss this method and the strategies to capture short-lived intermediate states with high spatial resolution on a time scale of ~ 20 ms to 1 second [2].  We have studied short-lived states during the process of bacterial translation; specifically during the phases of initiation, recycling and termination.

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNA^fMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Although comparisons of the structures of the 30S and 70S initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNA^fMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Here, using time-resolved cryogenic electron microscopy, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNA^fMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

REFERENCES

[1] Lu, Z., Shaikh, T.R., Barnard, D., Meng, X., Mohamed, H., Yassin, A., Mannella, C.A., Agrawal, R.K., Lu, T.-M., and Wagenknecht, T. (2009) Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy.  J. Struct. Biol. 168, 388-395.

[2 ]Frank, J. Time-resolved cryo-electron microscopy: Recent progress. J Struct Biol 200, 303-306 (2017).