Dynamical scattering in ice-embedded proteins in conventional and scanning transmission electron microscopy

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Cryogenic electron microscopy (Cryo-EM) relies on phase object (PO) and weak phase object (WPO) approximations for 3D macromolecular structure determination.
• Understanding image formation in transmission electron microscopy (TEM) is crucial for accurate reconstruction of protein complexes.

Purpose of the Study:

• To investigate the effects of multiple scattering and sample thickness on image formation in cryo-EM of tobacco mosaic virus (TMV).
• To compare conventional TEM simulations with ptychographic reconstructions using scanning transmission electron microscopy (STEM).

Main Methods:

• Dynamical multislice simulations of TMV specimens embedded in glass-like ice.
• Atomistic molecular dynamics simulations to model specimen structure.
• Ptychographic reconstruction analysis using STEM data.

Main Results:

• Single-slice models provide full frequency transfer up to 2.5 Å, with information transfer attenuating by 1.4 Å.
• Three slices are sufficient for information transfer up to 1.0 Å.
• Ptychographic reconstructions, unlike conventional TEM, do not require aberration introduction and allow post-acquisition correction, improving information transfer beyond 1.8 Å.

Conclusions:

• Multiple scattering and sample thickness are critical factors in cryo-EM image formation.
• Advanced simulation and reconstruction methods like ptychography enhance resolution and accuracy in structural determination of biological macromolecules.