Fully genetically encoded low-molecular-weight protein tags with defined shapes for direct molecular identification by cryo-electron tomography.

Cryo-electron tomography (cryo-ET) enables three-dimensional visualization of cells in near-native states, but direct identification of specific proteins in situ remains challenging due to crowded cellular environments and the low intrinsic contrast of most proteins smaller than ~500 kDa. Consequently, molecular identification often relies on indirect labeling strategies or bulky probes that can perturb native structures. Here we present a "shape-as-signal" strategy that uses fully genetically encoded protein tags with defined shapes as a molecular signal for direct identification by cryo-ET. We designed two single-chain, monomeric, low-molecular-weight tags: an extended V-shaped tag (62 kDa) and a compact triangular tag (85 kDa). Both adopt rigid geometries validated by cryo-electron microscopy and remain compatible with fluorescence microscopy when fused to fluorescent proteins. Their characteristic shapes are readily recognized and computationally detected in vitro. In cells, the V-shaped tag yields clear, non-disruptive signals at native locations. These results demonstrate that low-molecular-weight protein tags can be unambiguously detected and assigned in situ within crowded cellular environments. This single-step genetic tagging strategy enables seamless dual fluorescence and electron microscopy without exogenous probes, challenging the assumption that small protein tags are unsuitable for direct cryo-ET identification. More broadly, this approach establishes a scalable and minimally perturbative framework for visual proteomics and paves the way for multiplexed, shape-encoded molecular mapping in intact cells.