TomoNet: A streamlined cryoET software pipeline with automatic particle picking on flexible lattices

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 3D classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in one-, two- or three-dimensional arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other employing 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. ### Competing Interest Statement The authors have declared no competing interest.