An instrument for physical vapor deposition onto cryo-electron microscopy samples for microsecond time-resolved...

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) time resolution has advanced to the microsecond scale using laser flash melting.
• This advancement allows observation of protein dynamics crucial for function.
• Sample preparation for cryo-EM can be enhanced by altering particle environments during flash melting.

Purpose of the Study:

• To describe an apparatus for physical vapor deposition onto cryo-EM samples.
• To demonstrate the utility of this apparatus in optimizing laser flash melting experiments.
• To establish a new strategy for initiating protein dynamics in time-resolved cryo-EM.

Main Methods:

• Development and operation of a physical vapor deposition apparatus for cryo-EM samples.
• Laser flash melting and re-vitrification experiments.
• Determination of minimum silicon dioxide membrane thickness for sealing.

Main Results:

• The minimum thickness of silicon dioxide sealing membranes was determined to be just over two monolayers.
• It is possible to deposit reagents onto cryo-EM samples and induce mixing during flash melting.
• This method effectively removes preferred particle orientation by detaching particles from interfaces.

Conclusions:

• The developed apparatus facilitates microsecond time-resolved cryo-EM experiments.
• Physical vapor deposition offers a new strategy for initiating protein dynamics in time-resolved studies.
• The instrument design can serve as a foundation for an integrated platform for advanced cryo-EM applications.