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Single-particle cryo-electron microscopy (cryo-EM) is a technique for biomolecular structure reconstruction from vitrified samples containing many copies of a biomolecular complex (known as single particles) at random unknown 3D orientations and positions. Cryo-EM allows reconstructing multiple conformations of the complexes from images of the same sample, which usually requires many rounds of 2D and 3D classifications to disentangle and interpret the combined conformational, orientational, and translational heterogeneity. The elucidation of different conformations is the key to understand molecular mechanisms behind the biological functions of the complexes and the key to novel drug discovery. Continuous conformational heterogeneity, due to gradual conformational transitions giving raise to many intermediate conformational states of the complexes, is both an obstacle for high-resolution 3D reconstruction of the conformational states and an opportunity to obtain information about multiple coexisting conformational states at once. HEMNMA method, specifically developed for analyzing continuous conformational heterogeneity in cryo-EM, determines the conformation, orientation, and position of the complex in each single particle image by image analysis using normal modes (the motion directions simulated for a given atomic structure or EM map), which in turn allows determining the full conformational space of the complex but at the price of high computational cost. In this article, we present a new method, referred to as DeepHEMNMA, which speeds up HEMNMA by combining it with a residual neural network (ResNet) based deep learning approach. The performance of DeepHEMNMA is shown using synthetic and experimental single particle images.

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Understanding how structure and function meet to drive biological processes is progressively shifting the cryoEM field towards a more advanced analysis of macromolecular flexibility. Thanks to techniques such as single-particle analysis and electron tomography, it is possible to image a macromolecule in different states, information that can subsequently be extracted through advanced image-processing methods to build a richer approximation of a conformational landscape. However, the interoperability of all of these algorithms remains a challenging task that is left to users, preventing them from defining a single flexible workflow in which conformational information can be addressed by different algorithms. Therefore, in this work, a new framework integrated into Scipion is proposed called the Flexibility Hub. This framework automatically handles intercommunication between different heterogeneity software, simplifying the task of combining the software into workflows in which the quality and the amount of information extracted from flexibility analysis is maximized.