Cryo-electron microscopy (cryo-EM) has become a core technique for resolving high‑resolution protein three-dimensional structures. However, sample pre-treatment represents a major bottleneck restricting imaging quality. Common challenges include protein preferential orientation at the air-water interface, partial denaturation upon adsorption to carbon grids, particle aggregation, excessive background noise and uneven ice-layer thickness. These artefacts produce blurred particle projections, reduce valid particle counts and limit achievable resolution. Triglycine, applied as a small-molecular additive during cryo-EM sample pre-treatment, effectively mitigates these adverse effects and significantly improves imaging clarity of protein specimens.
Lacking bulky side-chain groups, triglycine features high water-solubility, low molecular weight and weak non-specific binding toward most protein surfaces. When supplemented into protein sample solutions before grid blotting and plunge freezing, triglycine molecules accumulate at the air-water interface without inserting into or disrupting native protein tertiary folds. It competes with target protein molecules for interfacial adsorption sites, alleviating protein denaturation triggered by direct contact with the hydrophobic air-water boundary. This suppresses the formation of partially unfolded protein particles, which are responsible for fuzzy particle images and heterogeneous particle conformations in micrographs.
Triglycine also modulates interfacial physicochemical properties to relieve particle aggregation. By adjusting surface tension and local ionic microenvironment, it reduces abnormal intermolecular hydrophobic and electrostatic attraction that causes protein clumping. More monodisperse individual protein particles are distributed evenly across thin ice layers. Well-separated particles decrease overlapping projections, increase the number of high-quality single-particle images available for subsequent reconstruction, and lower background interference originating from aggregated debris.
Benefiting from its small size, triglycine does not introduce prominent granular background noise under electron beam irradiation. Unlike large-sized protective polymers or crowding agents that generate hazy background signals and obscure protein features, triglycine molecules disperse uniformly within vitreous ice. During data collection, they produce negligible contrast interference, preserving genuine structural information of target proteins. Particles display sharper boundary contours, more distinguishable secondary-structure features and higher signal-to-noise ratio in raw micrographs, laying a solid foundation for high-resolution three‑dimensional reconstruction.
Nevertheless, concentration window must be strictly controlled in practical cryo-EM pre-treatment. Insufficient triglycine dosage cannot form effective interfacial competitive protection, so improvements remain limited. Excess triglycine elevates solution osmotic pressure, may alter protein solubility, and even induces subtle conformational shifts of sensitive macromolecular complexes. Optimised incubation time is also required to allow adequate interfacial redistribution before plunge freezing. Triglycine serves as an auxiliary additive rather than a universal remedy; it cannot rescue severely unstable or heavily degraded protein samples.
Triglycine acts as a functional additive for cryo-electron microscopy pre-treatment. It relieves protein denaturation at the air‑water interface, mitigates particle aggregation, maintains native particle dispersion, and avoids extra background noise. These combined effects enhance particle integrity and micrograph clarity, increasing the proportion of valid particles for single-particle analysis. It offers a convenient and practical sample-preparation strategy to support high-resolution structural determination of protein macromolecules.
