Rapid Production and Cryo-EM Structure Determination of GPCR-Nanodisc Complexes via Cell-Free Synthesis

G protein-coupled receptors (GPCRs) represent major therapeutic targets, yet cryo-EM structure determination of unliganded or antagonist-bound GPCRs remains severely bottlenecked by small receptor mass, detergent-induced instability, and multi-week cell-based production pipelines. Here, we combine intracellular loop 3 (ICL3) rigid fiducial engineering with a high-throughput, cell-free protein synthesis workflow to rapidly generate cryo-EM-ready GPCR–nanodisc assemblies. Using the eProtein Discovery™ digital microfluidics system, we performed automated nanoliter-scale screening of construct fusions and nanodisc lipid compositions, establishing that co-translational insertion into nanodiscs is strictly required for receptor solubilization and affinity purification. Direct scale-up of screening conditions yielded structural-grade protein within four days from gene assembly to grid vitrification. We demonstrate this workflow with a 3.38 Å cryo-EM structure of antagonist-bound human adenosine A _2A receptor (A _2A R)-β-lactamase fusion in complex with β-lactamase inhibitory protein II (BLIP-II). To demonstrate generalizability of protein quality, we extended this detergent-free co-translational pipeline to an expanded panel of 12 wild-type GPCRs spanning Classes A, B, and C, as well as six orphan receptors. All targets were purified with biophysical quality comparable to the A _2A R benchmark. By removing the reliance on cellular expression or agonist/G-protein stabilization and compressing construct assembly, nanodisc screening, biophysical quality control, and grid vitrification into a four-day turnaround, this detergent-free cell-free pipeline provides a rapid Design–Build–Test–Learn (DBTL) engine to accelerate structure-based drug discovery for challenging and unliganded GPCR targets.