High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction

Area of Science:

• Structural Biology
• Crystallography
• Electron Microscopy

Background:

• 3D electron diffraction is effective for determining protein structures.
• Producing suitable protein nanocrystals and minimizing solvent for electron diffraction remains a challenge.
• Focused ion beam (FIB) milling is one method to prepare protein crystals for electron diffraction.

Purpose of the Study:

• To introduce and evaluate cryo-sectioning of high pressure frozen protein crystals as a novel sample preparation technique for 3D electron diffraction.
• To assess the feasibility of obtaining high-resolution diffraction data from cryo-sectioned protein crystals.

Main Methods:

• Protein crystals were embedded in dextran and high pressure frozen.
• 150-200 nm thick cryo-sections were prepared.
• Electron diffraction data were collected from cryo-sectioned hen egg white lysozyme crystals.

Main Results:

• Complete diffraction data were obtained up to 2.9 Å resolution.
• The structure of lysozyme was solved by molecular replacement and refined.
• Cryo-sectioning successfully preserved protein structure for high-resolution analysis.

Conclusions:

• Cryo-sectioning is a viable new sample preparation technique for 3D electron diffraction of protein crystals.
• This method combines advantages of blotting and FIB-milling, offering controlled thickness and minimal solvent.
• The technique facilitates high-resolution structure determination by enabling analysis of numerous crystal orientations.