![]() Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). Evidence suggests that RNA aids nucleation of these pathological aggregates however, the mechanism has not been investigated at the level of atomic structure. In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. ![]() Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA. The structures illustrate more » homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The Legobody approach thus overcomes the current size limitations of cryo-EM analysis. ![]() The utility of the method more » is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The overall ensemble of ~120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). ![]()
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