An L-phenylalanine coating for holey carbon cryo-EM grids improves particle distribution

Abstract Electron cryomicroscopy (cryo-EM) enables macromolecular structure determination at near-atomic resolution, yet specimen preparation remains a critical bottleneck. In particular, the number of particles available within the ice-filled holes is often limiting, and simple surface strategies for increasing it remain few. Taking conceptual inspiration from hydrophobic natural surfaces, we coated holey carbon grids with the hydrophobic amino acid L-phenylalanine. The carbon support was first activated by UV-C irradiation and mild glow-discharge plasma treatment to facilitate L-phenylalanine attachment. X-ray photoelectron spectroscopy was used to characterise the elemental composition of the surfaces; the dilute coating lay below its detection limit, and functional evidence consistent with L-phenylalanine-mediated surface modification was instead obtained from a significant rise in the static water contact angle. Using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex as a model specimen, the coated grids yielded an approximately 2.3-fold increase in particles per micrograph compared with conventionally prepared grids, while two-dimensional classification indicated that specimen integrity was preserved. Because the coated and control grids differed in several steps of the preparation, this effect is attributed to the complete coating protocol rather than to L-phenylalanine alone. This facile and environmentally benign strategy offers a practical route to improving cryo-EM data collection efficiency.