Overcoming air-water interface-induced artifacts in Cryo-EM with protein nanocrates

Area of Science:

• Structural Biology
• Biochemistry
• Biophysics

Background:

• Cryo-electron microscopy (cryo-EM) sample preparation is susceptible to air-water interface artifacts.
• These artifacts include preferred orientation and molecular damage, hindering high-resolution structure determination.

Purpose of the Study:

• To introduce a novel method using protein shells called "nanocrates" to mitigate air-water interface issues in cryo-EM.
• To demonstrate the versatility and effectiveness of nanocrates across different protein types and structural challenges.

Main Methods:

• Encapsulation of target proteins within hydrophilic and stable nanocrates.
• Cryo-EM data acquisition and image processing for packaged proteins.
• 3D reconstruction of macromolecular structures prepared using the nanocrate method.

Main Results:

• Successful high-resolution cryo-EM structure determination of apoferritin (ApoF) using nanocrates.
• Overcoming preferred orientation issues for thyroglobulin (Tg) through nanocrate encapsulation.
• Determining the cryo-EM structure of 7,8-dihydroneopterin aldolase (DHNA), a protein previously uncharacterized by cryo-EM.

Conclusions:

• Nanocrates effectively prevent air-water interface-induced problems in cryo-EM sample preparation.
• The nanocrate method enables high-quality structural analysis of diverse proteins, including those with challenging properties.
• This technique advances cryo-EM by improving sample homogeneity and data quality for structural biology.