Integrating in situ single-particle cryo-electron microscopy with cryo-electron tomography for high-resolution structural biology in native cellular contexts.

Single-particle cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling atomic-resolution structure determination of purified macromolecular assemblies. Recent advances in in situ single-particle cryo-EM have extended this capability to near-atomic structural analysis directly within native cellular environments. However, major challenges remain because many cellular targets are low in abundance, structurally heterogeneous, and difficult to detect. In parallel, cryo-electron tomography (cryo-ET) combined with subtomogram averaging enables in situ visualization of macromolecular assemblies while preserving their three-dimensional cellular context, but limited throughput and resolution have constrained high-resolution analysis of rare or heterogeneous complexes. In this review, we discuss recent advances in sample preparation, data acquisition, image processing, and high-resolution refinement that improve the throughput, sensitivity, and resolution of in situ structural biology. We further highlight how integrating in situ single-particle cryo-EM with cryo-ET bridges cellular visualization and near-atomic structure determination, providing a scalable framework for investigating dynamic macromolecular assemblies directly in their native cellular context.