Effects of cryo-EM cooling on structural ensembles

Area of Science:

• Structural biology
• Biophysics
• Computational modeling

Background:

• Cryo-electron microscopy (cryo-EM) determines macromolecular structures at atomic resolution.
• Cryo-EM imaging requires rapid cooling of samples to cryogenic temperatures.
• The impact of this cooling process on the structural ensemble of macromolecules is not well understood.

Purpose of the Study:

• To quantify the effects of rapid cooling on the structural ensemble of macromolecules.
• To understand how cooling perturbs the native structure and influences observed heterogeneity.
• To provide a method for correcting or accounting for cooling-induced changes in cryo-EM data.

Main Methods:

• Combined continuum model calculations for temperature drop analysis.
• Molecular dynamics simulations of a ribosome complex during cooling.
• Kinetic modeling to analyze conformational changes and barrier crossings.

Main Results:

• Identified three primary effects contributing to the narrowing of structural ensembles during cooling: thermal contraction, reduced local motion, and equilibration to lower free-energy states.
• Observed that cooling allows overcoming free-energy barriers below 10 kJ/mol.
• These effects are expected to reduce B-factors in cryo-EM datasets.

Conclusions:

• Cooling significantly alters the structural ensemble of macromolecules, leading to a more homogeneous population.
• The study provides a quantitative framework to understand and potentially correct for cooling-induced artifacts in cryo-EM.
• This approach enables better interpretation of structural heterogeneity in room-temperature ensembles derived from cryo-EM structures.