Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets.

A simple method to determine transient conformations of biological molecules is described. The two reactants (e.g. protein complex and ligand) are mixed rapidly by the coalescence of spray droplets containing one component, with a thin, grid-supported aqueous film containing the other. The transient state is then trapped by rapid freezing, and investigated later by cryo-microscopy. Images of conformations associated with reaction times of 1-100 ms can be achieved by adjusting the delay between the droplet impact and freezing. The droplets (typically 1 micron in diameter) are propelled onto the grid by an atomizer spray. It is shown that the droplets impinging on the liquid film spread rapidly over its surface under the influence of surface tension, and only weakly disturb the underlying film, partially displacing its contents away from the point of impact. Experiments with sprayed salt solutions, using vesicles derived from erythrocytes as micro-osmometers, indicate that rapid mixing occurs both through the film and laterally, by diffusion. The spraying process does not produce any detectable concentration changes due to drying in either the droplets or the film, and the method is applicable to high-resolution imaging.