Near-atomic resolution reconstructions from in situ revitrified cryo samples

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Fast protein conformational dynamics are crucial for biological function.
• Observing these dynamics requires specialized techniques capable of capturing transient states.
• Conventional cryo-electron microscopy (cryo-EM) typically visualizes static structures.

Purpose of the Study:

• To evaluate the feasibility of obtaining high-resolution cryo-EM reconstructions from in situ revitrified samples.
• To assess the quality of reconstructions generated by this time-resolved cryo-EM method.
• To investigate the impact of in situ revitrification on particle orientation distributions.

Main Methods:

• Development and application of a microsecond time-resolved cryo-EM technique.
• Utilizing laser-induced local melting and rapid subsequent revitrification of cryo samples.
• Performing in situ revitrification experiments within the cryo-EM workflow.
• Image processing and reconstruction of trapped protein conformations.

Main Results:

• Achieved near-atomic resolution reconstructions from in situ revitrified cryo samples.
• Reconstruction quality was indistinguishable from conventional cryo-EM samples at comparable resolutions.
• Observed a more homogeneous angular distribution of particles after revitrification.

Conclusions:

• In situ revitrification is a viable method for time-resolved cryo-EM, yielding high-resolution structural data.
• This technique can potentially overcome preferred particle orientation issues in cryo-EM.
• Enables the study of fast protein dynamics and transient conformational states.