Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). 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 nanobodybinding protein A fragment fused to maltose binding protein and Fab-binding domains. 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 utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-angstrom 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-angstrom resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

Automated summary

The Legobody approach allows structure determination of small proteins using cryo-EM, by rigidly attaching a nanobody to two scaffolds for particle alignment. This method overcomes size limitations, demonstrated by obtaining high-resolution maps for the KDEL receptor and the RBD of SARS-CoV-2 spike protein.