Structural Studies of Macromolecular Assemblies Using Cryo-EM

Project Summary/Abstract The main objective of the proposed studies is the elucidation of fundamental processes of translation, translational regulation and translational quality control. To this end, single-particle cryo-electron microscopy, the technique pioneered in the PI's lab, is used in collaborations with world specialists on bacterial and

eukaryotic translation. We make use of two techniques of sample preparation, standard and time-resolved cryo-EM. In the standard application of cryo-EM, samples are pipetted onto the grid, excess liquid is removed by blotting, and the grid is then plunged into the cryogen. Since this procedure requires several seconds, it is

not possible to capture short-lived (less than 1000 millisecond) states of a molecule following a reaction. The alternative technique developed in this lab is time-resolved cryo-EM, whereby a reaction is started by mixing two components in a microfluidic chip, allowing them to react in a channel of defined, variable length (10 to

1000 ms), and then spraying the reaction products onto the grid as the latter in plunged into the cryogen. In this way, the kinetics of a reaction can be followed and, at the same time, intermediate states can be captured and visualized at high resolution. These two techniques are used to study the following processes: translation

initiation in E. coli and yeast, translation termination, recycling and quality control in mammalian, EMCV virus takeover of the host's ribosome. Another objective of the proposed studies is the exploration of a novel method of data analysis that seeks to generate a low-dimensional map of states existing in a continuum from a large

dataset of single-particle cryo-EM images of a biological macromolecule. Such a mapping can be used to determine the free-energy landscape of the molecule, containing information on the function-related conformational trajectories. This method will be applied in collaborations with leading experts to two

membrane-associated molecules with eminent biological and public health significance: rotary ATPase and Cystic Fibrosis trans-membrane conductance regulator (CFTR).