Evans, L.; Dingeldein, L.; Covino, R.; Gilles, M. A. T.; Thiede, E.; Cossio, P.

Cryo-electron microscopy (cryo-EM) experiments take 2D snapshots of individual proteins. In principle, these snapshots contain not only the main biomolecular conformations but also scarcely populated states and rare transitions between intermediates. This makes cryo-EM a powerful tool, not only for investigating the structure of biomolecules at high resolution but also for inferring the entire conformational ensemble distribution. Some recent works have reported conformational state populations by counting particle-images from cryo-EM. We wish to caution the community that these measurements are highly susceptible to noise and should not be relied upon as a precise estimate of the thermodynamic landscape of a biomolecule for understanding its biological function. Here, we demonstrate that the extremely noisy nature of cryo-EM images and uncertainty in the viewing orientations of biomolecules lead to ambiguities when assigning images to structures. If ignored, this ambiguity can introduce inherent bias when determining the populations of conformational states through individual particle assignment. We further show that modeling the conformational probability distribution using the entire image dataset mitigates these biases.