Comparing cryo-EM methods and molecular dynamics simulation to investigate heterogeneity in ligand-bound TRPV1.

Abstract

Cryogenic electron microscopy (cryo-EM) has emerged as a powerful method for resolving the structure of biological macromolecules. Recently, several computational methods have been developed to study the heterogeneity of molecules in single-particle cryo-EM. In this study, we analyze a publicly available dataset of TRPV1 using five such methods: 3DFlex, 3DVA, cryoDRGN, ManifoldEM, and Bayesian ensemble reweighting. We find significant heterogeneity, but each method produces different results, with some detecting only compositional or conformational heterogeneity. To compare these diverse results, we develop AnaVox to quantitatively determine agreement between heterogeneity methods. Furthermore, applying Bayesian ensemble reweighting combined with molecular dynamics simulations supports the presence of these rarer states within the sample. This study shows that although current methods reveal the presence of heterogeneity, their stochasticity and potential bias present challenges for their routine use. However, with future development, these tools will enable the use of cryo-EM data for quantitative biophysical investigations.