A Bayesian approach for extracting free energy profiles from cryo-electron microscopy experiments using a path collective variable

TL;DR

This paper introduces cryo-BIFE, a Bayesian method to extract free energy profiles from cryo-EM data, enabling insights into biomolecular conformational landscapes despite low SNR.

Contribution

The work presents a novel Bayesian framework using a path collective variable to recover free energy profiles from cryo-EM images, validated on synthetic and real data.

Findings

Successfully recovered free energy landscapes in synthetic systems.

Identified metastable states and activation barriers in a calcium channel.

Demonstrated the method's effectiveness on real cryo-EM particles.

Abstract

Cryo-electron microscopy (cryo-EM) extracts single-particle density projections of individual biomolecules. Although cryo-EM is widely used for 3D reconstruction, due to its single-particle nature, it has the potential to provide information about the biomolecule's conformational variability and underlying free energy landscape. However, treating cryo-EM as a single-molecule technique is challenging because of the low signal-to-noise ratio (SNR) in the individual particles. In this work, we developed the cryo-BIFE method, cryo-EM Bayesian Inference of Free Energy profiles, that uses a path collective variable to extract free energy profiles and their uncertainties from cryo-EM images. We tested the framework over several synthetic systems, where we controlled the imaging parameters and conditions. We found that for realistic cryo-EM environments and relevant biomolecular systems, it is possible to recover the underlying free energy, with the pose accuracy and SNR as crucial determinants. Then, we used the method to study the conformational transitions of a calcium-activated channel with real cryo-EM particles. Interestingly, we recover the most probable conformation (used to generate a high resolution reconstruction of the calcium-bound state), and we find two additional meta-stable states, one which corresponds to the calcium-unbound conformation. As expected for turnover transitions within the same sample, the activation barriers are of the order of a couple $k_BT$. Extracting free energy profiles from cryo-EM will enable a more complete characterization of the thermodynamic ensemble of biomolecules.