Modular microfluidics enables kinetic insight from time-resolved cryo-EM

Biochemical reactions are driven by highly dynamic conformational changes in biological macromolecules, typically occurring on microsecond to millisecond timescales. Understanding these changes is crucial for elucidating reaction mechanisms, but experimental challenges exist in isolating and characterizing functionally relevant intermediates. Cryo-electron microscopy (cryo-EM) is a powerful technique for determining macromolecular structures, but traditional methods are limited in their ability to resolve short-lived intermediates. Previous time-resolved cryo-EM (trEM) approaches have been constrained by slow kinetics, temperature sensitivity, or inability to enrich short-lived intermediates. The development of a general, time-resolved sample preparation method for cryo-EM necessitates a miniaturized mixer and bioreactor capable of rapidly initiating and synchronizing biochemical reactions and applying the sample to a cryo-EM grid blot-free, faster than the lifetime of the structures of interest. While existing microfluidic approaches offer promise, significant technical challenges remain in terms of design complexity, reproducibility, mixing efficiency on millisecond timescales, and reliable blot-free sample application. This paper aims to address these challenges.

Numerous studies have highlighted the importance of dynamic conformational changes in protein function. Cryo-EM and single-particle analysis have become widely used for structural determination, offering advantages in near-native sample conditions. However, standard cryo-EM workflows cannot directly identify functionally relevant states or elucidate the temporal sequence of structural transitions. Previous attempts at time-resolved cryo-EM have yielded some success, primarily with techniques like cryo-electron crystallography, which is limited to crystalline samples, and methods that rely on slow kinetics or significant temperature sensitivity. Several groups have explored microfluidic-based approaches for time-resolved cryo-EM, showing the feasibility of rapid mixing and sample delivery, but challenges persist in terms of device complexity, reproducibility, and achieving reliable mixing and vitrification on relevant timescales.

This study presents an integrated trEM workflow based on a modular microfluidic platform. The platform features an in situ 3D mixer for efficient and rapid mixing of reactants (validated by confocal microscopy of fluorescent dyes, achieving near-perfect mixing in 3.1 ms with seven mixing junctions and a flow rate of 333 µl/min per channel). A gas-assisted nozzle produces a fine sample aerosol, enabling blot-free sample application onto cryo-EM grids. A servo motor controls the rapid plunging of the grid into liquid ethane for vitrification (optimized plunging speed of 100 rpm, 8mm distance from nozzle, resulting in an integration time of 2.7 ms and a total time from mixing to vitrification of 16ms, overall dead time of around 30 ms). The system's modular design promotes reproducibility and adaptability to different microfluidic devices and experimental needs. The method was validated using several commercially available cryo-EM grids (holey Quantifoil grids performing best) and various biological samples, including apoferritin, CSN5H138A-SCF-N8Skp2/Cks1 complex, and 20S proteasomes. Tomography demonstrated significantly more uniform protein distribution compared to standard methods. Single-particle analysis confirmed the ability to obtain high-resolution structures comparable to those obtained using standard preparation methods. RecA filament growth was used as a model system to assess the time resolution and accuracy of the trEM workflow. RecA was mixed with single-stranded DNA and ATPγS using the microfluidic device and filament lengths were measured in cryo-EM micrographs. Filament length distribution from the microfluidic preparation displayed a much narrower distribution than manual methods. The study also investigated potential sources of error, such as reactant adsorption to the PDMS microfluidic channels and residence time distribution due to fluid dynamics. Computational fluid dynamics simulations were performed to model and predict residence time distribution. A time-course of RecA filament growth reactions was conducted across a range of timescales (10-1000 ms) using microfluidic devices with varying channel lengths to demonstrate the system's ability to resolve pre-steady-state kinetic intermediates.

The study demonstrates the successful development and validation of a modular and versatile time-resolved cryo-EM (trEM) workflow. The microfluidic device achieves near-perfect mixing in under 3 milliseconds. Blot-free sample vitrification via gas-assisted spraying resulted in superior sample integrity and uniform protein distribution across the grid. High-resolution structures were obtained for several model proteins, including apoferritin (2.77 Å resolution). The trEM method is capable of capturing pre-steady-state reaction intermediates, as shown by the time-resolved analysis of RecA filament growth kinetics. This analysis demonstrated close agreement between structural and biophysical measurements of RecA filament growth rates (~20 nt/s). The experiments also revealed sources of error, such as reactant adsorption to the PDMS and residence time distribution from laminar flow within the microfluidic channels. Computational fluid dynamics simulations helped predict and understand the residence time distribution which provides essential guidance for future work. High-quality cryo-EM data was acquired and analysed for the model systems. A detailed characterisation of the experimental system enables reproducible measurements.

The trEM method addresses significant limitations of traditional cryo-EM sample preparation for time-resolved studies. The ability to capture short-lived intermediates and analyze pre-steady-state reaction kinetics opens new avenues for investigating dynamic molecular processes. The findings are relevant to understanding fundamental biochemical mechanisms and studying other complex systems. The lack of need for specific labels facilitates the holistic study of structural transitions without bias. The method’s modularity and relative simplicity enhance its accessibility and applicability in various biological research settings. The detailed characterization of potential sources of error provides a roadmap for further refinement and optimization. Future applications of this methodology include studying enzymatic mechanisms, protein-protein interactions, and other dynamic biological processes.

This study presents a significant advancement in time-resolved cryo-EM, providing a robust and versatile platform for capturing short-lived reaction intermediates. The modular microfluidic design, gas-assisted sample application, and comprehensive characterization of the system's performance lead to high-quality data suitable for high-resolution structural analysis and kinetic studies. The successful application to the RecA filament growth model system validates the method's capability to resolve pre-steady-state kinetics and provides a strong foundation for future investigations into a wide range of dynamic biological processes. Future work will focus on reducing sample consumption, improving time resolution through advanced microfluidic designs and minimizing the influence of residence time distribution effects.

While the trEM method significantly improves time resolution in cryo-EM, certain limitations remain. Reactant adsorption to the PDMS microfluidic channels and residence time distribution effects can introduce errors in time measurements. Reducing the overall dead time and improving the accuracy of synchronization remain challenges for future optimization. For complex systems with multiple interacting species and diverse conformational states, advanced image analysis techniques will be required to reliably resolve individual intermediates and their populations during the course of a reaction. The presented method may not be fully applicable to reactions with extremely fast kinetics, requiring further development of high-speed mixing techniques.