TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices

Abstract

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and three-dimensional (3D) classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in 1D, 2D, or 3D arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other using deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures. Impact StatementCryogenic electron tomography (cryoET) has become a powerful approach to visualize the organization and high-resolution structures of biological complexes in their native environment. Subtomogram averaging (STA) of hundreds of thousands of particles (i.e., subtomograms) is necessary to obtain near-atomic resolution structures for each such complex. While abundant biological complexes often cluster in arrays that manifest as one to three-dimensional lattices, flexibility and imperfection of such lattices pose challenges for efficient and accurate particle picking. To overcome these challenges and to meet the growing demand for efficient data processing and management in the cryoET and STA workflow, we have developed TomoNet, a user-friendly software package with a modern graphical user interface that allows users to execute the entire data processing pipeline seamlessly with the integration of commonly used software packages. TomoNet addresses the particlepicking challenge with two solutions, one based on geometric template matching and the other using artificial intelligence. Applications of TomoNet to three representative datasets demonstrate its capability for highresolution structure determination of biological complexes on flexible and imperfect lattices.