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.
ARCHER: Amortized cross-specimen pose estimation for cryo-electron microscopy
Pritzker School of Molecular Engineering, University of Chicago（芝加哥大学普利兹克分子工程学院）；Department of Computer Science, Rensselaer Polytechnic Institute（伦斯勒理工学院计算机科学系）
PERC: a suite of software tools for the curation of cryoEM data with application to simulation, modelling and machine learning
The Alan Turing Institute（阿兰·图灵研究所）；Science & Technology Facilities Council（科学与技术设施委员会）；Rosalind Franklin Institute（罗莎琳德·富兰克林研究所）；University of Oxford（牛津大学）；Diamond Light Source（钻石光源）
