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.

Cryo-electron tomography (cryo-ET) is a powerful technique that can provide unprecedented insight into protein-protein interactions and molecular machinery in a near-native state. The adoption of cryo-ET by life science research groups is hampered by the challenges associated with cryo-ET sample preparation. The current sample preparation process has many steps at which ice contamination may occur to negatively affect the final sample and data quality. A survey was conducted to better understand the effects and impact of ice contamination to the cryo-ET outcome. Over 80 cryo-electron microscopy users worldwide participated in our survey. The results are presented in this article. We furthermore discussed the currently available solutions that can alleviate the ice contamination problems to increase the sample yield and cryo-ET data output.