On-grid and in-flow mixing for time-resolved cryo-EM

Area of Science:

• Structural Biology
• Biochemistry
• Biophysics

Background:

• Time-resolved cryo-electron microscopy (TrEM) is an emerging technique for observing rapid biological processes.
• Current TrEM methods have limitations in achieving short time delays and require further validation against established biochemical assays.

Purpose of the Study:

• To compare different approaches for time-resolved cryo-electron microscopy (TrEM) sample preparation.
• To validate TrEM kinetic data by comparing it with established biochemical measurements.
• To investigate the reaction kinetics of the skeletal actomyosin S1 complex with ATP.

Main Methods:

• Utilized time-resolved cryo-electron microscopy (TrEM) with varying delay times (7-700 ms) between mixing and vitrification.
• Employed on-grid and in-flow mixing techniques for sample preparation.
• Analyzed cryo-EM data through classification to extract kinetic information.

Main Results:

• Derived kinetic data from TrEM that aligns with previous biochemical studies of actomyosin S1 complex.
• Observed fast dissociation, low steady-state hydrolysis occupancy, and subsequent rebinding of ATP.
• Noted a reduced rebinding effect with on-grid mixing and potential influence of air-water interface interactions.
• Identified temporal spread in in-flow mixing, particularly at longer delay times.

Conclusions:

• TrEM is a powerful tool for studying protein dynamics on millisecond timescales.
• The study highlights challenges in TrEM, including temporal spread and air-water interface effects.
• Further development of TrEM techniques is needed to overcome current limitations and expand its applications.