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 a 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 individual particles. In this work, we propose the cryo-BIFE method (cryo-EM Bayesian Inference of Free-Energy profiles), which uses a path collective variable to extract free-energy profiles and their uncertainties from cryo-EM images. We test the framework on several synthetic systems where the imaging parameters and conditions were controlled. 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. We then use the method to study the conformational transitions of a calcium-activated channel with real cryo-EM particles. Interestingly, we recover not only the most probable conformation (used to generate a high-resolution reconstruction of the calcium-bound state) but also a metastable state that corresponds to the calcium-unbound conformation. As expected for turnover transitions within the same sample, the activation barriers are on the order of k_BT. We expect our tool for extracting free-energy profiles from cryo-EM images to enable more complete characterization of the thermodynamic ensemble of biomolecules.
Highlights
Cryo-electron microscopy extracts single-particle density projections of individual biomolecules
In cryo-electron microscopy experiments a biomolecular sample is immersified in vitrified ice
To understand the effects of the physical parameters for recovering free-energy profiles with cryo-BIFE, we designed several control systems where the projections are generated synthetically following the ideas of Ref.[43]
Summary
Cryo-electron microscopy (cryo-EM) extracts single-particle density projections of individual biomolecules. Cryo-EM is widely used for 3D reconstruction, due to its single-particle nature it has the potential to provide information about a biomolecule’s conformational variability and underlying free-energy landscape. We expect our tool for extracting free-energy profiles from cryo-EM images to enable more complete characterization of the thermodynamic ensemble of biomolecules. One can characterize relevant biophysical properties, such as the free-energy landscape, activation barriers, transition states, and transition paths between conformations. This can provide essential clues to biomolecular function[9]. Several methods have been developed to extract 3D density maps of heterogeneous biomolecules using cryo-EM These methods can be divided into two types: discrete-state or continuous-state methods. It is not trivial to determine if the system’s conformational changes are best modeled by a discrete or continuous set of s tates[13]
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