Preparation of High-Temperature Sample Grids for Cryo-EM

Area of Science:

• Structural Biology
• Biochemistry
• Cryo-Electron Microscopy

Background:

• Conventional cryo-electron microscopy (cryo-EM) sample preparation occurs at low temperatures (e.g., 4 °C), potentially yielding non-functional protein structures.
• Proteins from thermophilic archaea may exhibit altered structures and functions at standard low-temperature preparation conditions.
• The functional relevance of protein structures solved at low temperatures is questionable, particularly for extremophiles.

Purpose of the Study:

• To develop and detail a protocol for preparing cryo-EM sample grids at elevated temperatures (up to 70 °C).
• To demonstrate that high-temperature sample preparation yields functionally relevant and temperature-dependent protein structures.
• To provide a method for obtaining accurate structural insights into proteins, especially those from thermophilic organisms.

Main Methods:

• Modification of a standard vitrification apparatus with an additional centrifuge tube for high-temperature incubation.
• Sample incubation at elevated temperatures, exemplified by 55 °C, prior to grid plunging.
• Optimization of procedures to minimize vapor condensation and ensure thin ice layer formation on cryo-EM grids.

Main Results:

• Successful preparation of cryo-EM sample grids at temperatures up to 55 °C.
• Demonstration that protein structures obtained from high-temperature prepared samples are functionally relevant.
• Evidence of temperature-dependent structural variations in proteins prepared using the novel method.

Conclusions:

• High-temperature sample preparation is crucial for determining the functionally relevant structures of proteins, particularly thermophilic archaea.
• The developed protocol enables accurate cryo-EM structural analysis at temperatures reflecting native conditions.
• This methodology advances structural biology by providing a pathway to study temperature-dependent protein conformations.