Li, Y.; Zhang, Y.; Wu, C.; Zhu, C.; Xiong, Y.

Resolving macromolecular structures within their native cellular environment is essential for connecting molecular architecture to physiological function, yet the field lacks accessible, high-throughput methods for generating suitable specimens from cells. Here, we introduce cryoGO (on-Grid Opening cryo-electron microscopy), a simple, rapid, and scalable strategy that mechanically opens cells directly on EM grids to produce cell-derived specimens for high-resolution single-particle cryo-EM. This approach enables near-atomic structure determination of diverse endogenous macromolecular assemblies spanning a broad molecular-weight range. We show that cryoGO captures the rich ribosomal conformational and compositional landscape while preserving physiological state distributions and aspects of spatial heterogeneity originating from cells. Furthermore, this rapid workflow enables time-resolved structural profiling, capturing both long-term cellular adaptation and acute remodeling on timescales of seconds. Compatible with standard cryo-EM infrastructure and diverse biological samples, while requiring only small numbers of cells, cryoGO lowers the technical barrier to high-resolution native structural biology.