Invited:
DIRECT CORRELATION BETWEEN STRUCTURE AND FUNCTION

Cryo-EM is unique in its ability to elucidate the molecular architecture of life close to native conditions in complex settings. Cryo-EM sample-preparation methods offer great flexibility in a selection of preparation conditions (e.g., additives, temperature). As result, molecular machines in action are salient targets for structure-function Cryo-EM studies.

Notably, Cryo-EM is in its essence a single molecule technique, where signals are collected from individual molecules. However, traditionally image processing of Cryo-EM data requires averaging signals from a large set of molecules, leading to low noise 3D structures at the expense of loss of information on the ensemble. While different conformations can be discerned by these approaches, it is hard to quantify the relative amounts of different conformations in a sample solely by image processing. Moreover, identification of small ligands (e.g. ADP or ATP), which is crucial for understanding the biochemical state of a macromolecule, is challenging.

Here I will discuss a novel approach for combining single molecule localization microscopy with Cryo-EM. In addition, I will present test cases demonstrating the accuracy of the analytical capabilities of Cryo-EM in determining the relative amounts of different conformations in a sample. Altogether, these methods will enable direct correlation between the conformations found in the microscope, and the biochemical state of the macromolecule. These advances are utilized in our lab in order to elucidate the reaction cycle of molecular machines, in order to cast the basic dogma of structural biology in a statistical framework.