Studying Kinetics by Counting Particles in Time-Resolved Cryo-EM

In time-resolved cryo-EM, the progress of a reaction, started by mixing of two components, can be followed over a period of time by stopping the reaction and imaging the samples at defined intervals At each time point there will be a mixture of the two starting components, transient intermediate products, and end product. Most reactions of biological molecular machines of interest are completed within a few hundred milliseconds, necessitating special technology for fast mixing, reacting over a defined period of time, and depositing the reaction product on the EM grid. We have demonstrated that with a mixing/spraying microfluidic device previously developed in a collaboration between researchers at the Wadsworth Center and RPI [3], high-resolution reconstructions (in the 3- range) of short-lived intermediates can now be routinely obtained Thus, we can now achieve both spatial and temporal resolution in following the progress of a reaction. Importantly, kinetic data are obtained implicitly as a byproduct of classification: by simply counting the number of particles in each class (or subpopulation) for each time point captured. In principle, therefore, time-resolved cryo-EM is able to measure the kinetics of a macromolecular reaction by a sequence of experiments with microfluidic chips designed for a series of time points.