On-grid enrichment strategies in cryo-EM: affinity-functionalized supports for low-abundance and challenging targets

Cryo-electron microscopy (cryo-EM) has become a leading method for high-resolution structural analysis of biological macromolecules. Despite major advances in instrumentation and image processing, specimen preparation remains a critical bottleneck, particularly for membrane proteins, endogenous assemblies, chromatin-associated complexes and transient signaling intermediates. These targets are often available only in limited quantities and are susceptible to denaturation, dissociation and preferred orientation during conventional grid preparation. Affinity-functionalized grids have emerged as a powerful specimen-preparation strategy that enables selective on-grid capture, enrichment and purification prior to vitrification, thereby increasing the local particle concentration, improving particle recovery and, in some implementations, reducing exposure to the air-water interface. Here, we review affinity-functionalized and engineered cryo-EM supports as a unified class of specimen-preparation technologies and compare the major validated platforms, including nickel-nitrilotriacetic acid (Ni-NTA) lipid monolayers, streptavidin affinity grids, antibody-based supports, graphene-derived substrates and magnetic nanoparticle-based enrichment systems. We discuss their strengths, limitations and applications, as well as emerging developments in programmable capture chemistries, automated vitrification workflows and in situ structural biology. Collectively, these technologies are transforming the cryo-EM support from a passive substrate into an active biochemical interface, expanding the range of biological systems accessible to structural investigation.