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bioRxiv · 10.1101/2021.08.09.455715

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

Wu, X.·Rapoport, T. A.
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Abstract

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 nanobody-binding 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 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[A] overall resolution with density sufficient for de novo model building, and for the 22 kDa RBD of SARS-CoV2 spike protein, resulting in a map at 3.6 [A] resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

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Wu, X., Rapoport, T. A.. 2021-08-10. Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies). https://doi.org/10.1101/2021.08.09.455715

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