Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

TLDR

The authors propose using semi‑synthetic antibodies against the engineered BRIL protein as universal fiducial markers for single‑particle cryo‑EM of membrane proteins. They generate these antibodies through customized phage display selections against BRIL, then fuse BRIL to loops or termini of diverse GPCRs, ion channels, receptors, and transporters, enabling the antibodies to bind without perturbing protein structure. The antibodies bind BRIL‑fused membrane proteins across the tested classes, as confirmed by crystal structure of a BRIL–sAB complex, negative‑stain and cryo‑EM data, and a cryo‑EM structure of an sAB‑bound nicotinic acetylcholine receptor, demonstrating markedly improved particle properties and cryo‑EM outcomes for challenging membrane proteins.

Abstract

Abstract We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.