Programmable gold nanoparticle conjugates enable precise AMPA receptor localization at brain synapses by cryo-ET

Cryo-electron tomography (cryo-ET) is a powerful approach to image cells, organelles, viruses and molecular assemblies. However, resolving smaller complexes and molecules is challenging and presents a roadblock to the broad application of cryo-ET. Gold nanoparticles (AuNPs) have been used to localize otherwise unidentifiable molecules and complexes in tomograms but are typically tethered to the target of interest via a flexible linker. As a result, the position of the AuNP does not directly define the location of the target. Here we attach AuNPs to Fab antibody fragments via cysteine residues on defined elements of secondary structure and show that the AuNPs are rigidly bound to the Fab. We use these Fab-AuNP conjugates to locate AMPA receptors and probe their conformation upon desensitization with sub nanometer resolution in tomograms derived from brain slice lamella. By harnessing small, homogeneous AuNPs with precise conjugation to a Fab, we have developed reagents that enable direct investigation of conformational states of previously unidentifiable biomolecules and complexes in tomograms.