Single-particle cryogenic electron microscopy (cryo-EM), recognized by the award of the 2017 Nobel Prize in Chemistry to Dr. Joachim Frank of our research team, uses electron microscopy (EM) of specimens of biological molecules embedded in vitreous ice via rapid cryogenic cooling. Time-resolved cryo-EM (TRCEM), an expansion of cryo-EM in which specimens are examined resulting from reactions occurring for a series of controlled time durations, allows studies of short-lived intermediate states of biomolecular events and has recently been applied successfully to investigate several bacterial translation systems with reaction times ranging between 20 and 600 ms. TRCEM studies at higher time resolutions, however, have not been possible as existing specimen preparation methods preclude reaction times below 20 ms from being achieved. This capability gap is highly limiting, because there are a large number of ion channels and receptors implicated in human diseases with activation and gating times on the order of 1 ms or less. In addition, existing cryo-EM specimen preparation methods use devices that are each hardwired for a particular reaction time, which is inefficient and may cause inconsistencies in specimen quality. We will develop a microfluidic cryo-EM specimen preparation system, termed the High Uniformity and Resolution Reaction Intermediates (HURRI) system, to address these challenges. The system consists of a microsprayer or a line array of microsprayers in combination with a uniform reaction control and spray cryocooling (in short, react-cryocool) unit. The microsprayer rapidly mixes two reactants at a low flow rate, atomizes the mixture into monodisperse droplets, and delivers and deposits the droplets onto an EM grid. In the react-cryocool unit, the grid is rapidly transferred into a nearby cryogen spray, en route to which reactant molecules react for a short time independent of their on-grid position until being vitrified. Combining the microsprayer and react-cryocool technologies minimizes the length of the overall reaction zone as well as variations in the reaction time experienced by different reactant molecules. Thus, HURRI will be capable of generating specimens with submillisecond reaction times at high resolution and low dispersity. In addition, the reaction time will be tunable in that a given HURRI instrument will, without requiring modifications of any physical components, allow generation of specimens at a series of reaction times needed for a study. The reactant flow rates at which HURRI operates will also be one order of magnitude lower than those used in existing specimen preparation methods, thereby reducing the use of biological material. As such, the HURRI technology will enable highly efficient TRCEM studies of fast-reacting biological systems at an unprecedented high time resolution. This potentially transformative utility of HURRI will be demonstrated with a TRCEM case study of type 1 ryanodine receptor/calcium release channels (RyR1), the largest known ion channel that is required for numerous critical cellular functions.

Single-particle cryogenic electron microscopy (cryo-EM) is electron microscopy (EM) of biological molecules embedded in vitreous ice specimens. Time-resolved cryo-EM examines specimens that reflect biomolecular reactions occurring for a series of controlled time durations. We will develop a technology for preparing cryo-EM specimens with submillisecond reaction times to enable time-resolved cryo-EM studies of short-lived intermediate states of biomolecular systems.