Hierarchical autoclassification of cryo-EM samples and macromolecular energy landscape determination

Cryo-electron microscopy using single particle analysis is a powerful technique for obtaining 3D reconstructions of macromolecules in near native conditions. One of its major advances is its capacity to reveal conformations of dynamic molecular complexes. Most popular and successful current approaches to analyzing heterogeneous complexes are founded on Bayesian inference. However, these 3D classification methods require the tuning of specific parameters by the user and the use of complicated 3D re-classification procedures for samples affected by extensive heterogeneity. Thus, the success of these approaches highly depends on the user experience. We introduce a robust approach to identify many different conformations presented in a cryo-EM dataset based on Bayesian inference through Relion classification methods that does not require tuning of parameters and reclassification strategies.

The algorithm allows both 2D and 3D classification and is based on a hierarchical clustering approach that runs automatically without requiring typical inputs, such as the number of conformations present in the dataset or the required classification iterations. This approach is applied to robustly determine the energy landscapes of macromolecules.

We tested the performance of the methods proposed here using four different datasets, comprising structurally homogeneous and highly heterogeneous cases. In all cases, the approach provided excellent results. The routines are publicly available as part of the CryoMethods plugin included in the Scipion package.

Our results show that the proposed method can be used to align and classify homogeneous and heterogeneous datasets without requiring previous alignment information or any prior knowledge about the number of co-existing conformations. The approach can be used for both 2D and 3D autoclassification and only requires an initial volume. In addition, the approach is robust to the "attractor" problem providing many different conformations/views for samples affected by extensive heterogeneity. The obtained 3D classes can render high resolution 3D structures, while the obtained energy landscapes can be used to determine structural trajectories.