Could Egg White Lysozyme be Solved by Single Particle Cryo-EM?

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• High-end cryogenic transmission electron microscopes (cryo-EM) and advanced algorithms enable 3D structure determination of small macromolecules.
• However, routine structure determination for proteins under 100 kDa remains challenging due to sample characteristics and hardware limitations.

Purpose of the Study:

• To develop and validate a cryo-EM data simulator for assessing structure determination of small macromolecules.
• To evaluate the impact of phase plates on high-resolution 3D reconstruction of small proteins.

Main Methods:

• Simulated single-particle cryo-EM data sets using realistic parameters for ice layer, dose, detector, and beam characteristics.
• Calculated reference data for human apo-ferritin and processed simulated data.
• Generated simulated data for hen egg white lysozyme (14 kDa) with and without an ideal phase plate (PP).

Main Results:

• Simulated data for apo-ferritin yielded a 1.86 Å resolution map, comparable to experimental results.
• High-resolution 3D reconstruction of 14 kDa hen egg white lysozyme was not achieved without a phase plate.
• Using an ideal phase plate, a 2.78 Å resolution density map was obtained for hen egg white lysozyme.

Conclusions:

• The developed cryo-EM simulator aids in understanding and overcoming limitations for small macromolecule structure determination.
• Phase plates are crucial for achieving high-resolution cryo-EM structures of proteins smaller than 100 kDa.
• This work can help push the size limits of cryo-EM for routine structure determination.