Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins

TLDR

Cryo‑EM of membrane proteins often requires antibody fragments to aid alignment, yet existing nanobodies add only ~15 kDa, limiting their use as fiducials. The authors created NabFab, a synthetic Fab that rigidly binds the conserved scaffold of nanobodies to serve as a universal fiducial for cryo‑EM. They validated NabFab by solving two high‑resolution cryo‑EM structures of membrane proteins complexed with specific nanobodies and NabFab. Because NabFab’s epitope can be incorporated into any nanobody scaffold, it enables rapid structure determination of a wide range of nanobody–protein complexes.

Abstract

Significance Structural studies of membrane proteins by cryogenic electron microscopy (cryo-EM) often require antibody fragments (Fabs) to facilitate particle alignments and achieve high resolution. While conformational nanobodies have been developed to lock specific states of many membrane proteins, they only add 15 kDa of mass to the complex. We developed a synthetic Fab (NabFab) that rigidly binds the conserved scaffold of nanobodies, providing a universally applicable fiducial for cryo-EM studies of protein–nanobody complexes. We demonstrate the concept by determining two high-resolution structures of membrane proteins bound to specific nanobodies and NabFab. As the structural epitope for NabFab can be incorporated into the scaffold of virtually any nanobody, this raises the prospect of facile structure determination of many nanobody–protein complexes.