Super-Resolution Processing Enables In Situ Subtomogram Averaging Beyond the Nyquist Limit

In situ subtomogram averaging (STA) for high-resolution structure determination requires fine pixel sampling, forcing a trade-off between achievable resolution and the field of view (FOV) recorded in each tomogram. Electron-event representation (EER) data can be rendered at half the physical pixel size, but whether super-resolution processing can recover useful information beyond the physical Nyquist limit from noisy in situ data remains unclear. Here, we show that super-resolution processing of EER data recovers biologically interpretable information beyond a physical Nyquist limit of 4.78 A in two in situ datasets. STA of unidentified virus particles in a Drosophila melanogaster larva yielded a 3.42 A inner capsid reconstruction, enabling de novo model building and identification of the particles as Drosophila A virus. In situ STA of ribosomes in Saccharomyces cerevisiae reached 3.18 A, with local resolution extending to 2.78 A. EER acquisition therefore provides greater flexibility in choosing the acquisition magnification: existing datasets can be re-rendered at the super-resolution pixel size when physical sampling becomes limiting, while lower-magnification acquisition can be considered when a larger FOV improves target capture or cellular context.