Towards light-coupled sample preparation for time-resolved cryoEM studies

Area of Science:

• Structural Biology
• Biophysics
• Cell Biology

Background:

• Understanding protein dynamics is crucial for elucidating biological functions.
• Static structures offer limited insight into transient conformational changes during reactions.
• Capturing sequential states of macromolecular complexes is essential for functional analysis.

Purpose of the Study:

• To develop and validate a framework for time-resolved cryo-electron tomography (cryoET).
• To enable the visualization of rapid molecular events within their native cellular context.
• To study dynamic conformational changes in protein complexes at millisecond timescales.

Main Methods:

• Development of modular tools for optical excitation, on-grid characterization, and rapid vitrification.
• Application of a femtosecond-pulsed laser coupled to a Vitrobot for controlled sample preparation.
• Utilizing DMNB-caged serine as a trigger and UV-Vis spectroscopy and GC×GC-MS for characterization.
• Time-resolved cryo-electron tomography (cryoET) for in situ structural analysis.

Main Results:

• Established a proof-of-principle for millisecond time-resolved cryoET.
• Achieved reproducible reaction-to-vitrification delays of approximately 150 milliseconds.
• Successfully captured intact E. coli minicells with preserved chemotaxis arrays.
• Demonstrated the framework's capability for in situ structural analysis of dynamic processes.

Conclusions:

• The integrated approach provides a robust and generalizable framework for time-resolved cryoET.
• This method facilitates the study of transient conformational states in native cellular environments.
• Lays the groundwork for future investigations into dynamic biological processes at unprecedented temporal resolution.