“…Downstream subtomogram averaging (STA) (Galaz-Montoya and Ludtke, 2017;Förster, 2022) can yield structures at subnanometer (Schur et al, 2013) and near-atomic (Schur et al, 2016) resolutions, even for complexes within cells (Tegunov et al, 2021). While STA was first demonstrated nearly 30 years ago (Walz et al, 1997) using ad hoc scripts (Nicastro et al, 2006), the first semi-automated pipelines for STA started emerging only about a decade ago (Hrabe et al, 2012;Castaño-Díez et al, 2012;Galaz-Montoya et al, 2015), and have become increasingly sophisticated and efficient (Galaz-Montoya et al, 2016;Bharat and Scheres, 2016;Castaño-Díez et al, 2017;Himes and Zhang, 2018;Fernandez and Li, 2021;Zivanov et al, 2022;Burt et al, 2024), with the field rapidly taking large strides towards full automation for some specimens (Chen et al, 2019;Ni et al, 2022;Balyschew et al, 2023). Clearly, these advancements have positioned STA at the forefront of high-resolution structural biology in situ.…”