PT  - JOURNAL ARTICLE
AU  - Xin Zhang
AU  - Rachel M. Johnson
AU  - Ieva Drulyte
AU  - Lingbo Yu
AU  - Abhay Kotecha
AU  - Radostin Danev
AU  - Denise Wootten
AU  - Patrick M. Sexton
AU  - Matthew J. Belousoff
TI  - Evolving cryo-EM structural approaches for GPCR drug discovery
AID  - 10.1101/2021.01.11.426276
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.01.11.426276
4099  - http://biorxiv.org/content/early/2021/01/12/2021.01.11.426276.short
4100  - http://biorxiv.org/content/early/2021/01/12/2021.01.11.426276.full
AB  - G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.Competing Interest StatementThe authors have declared no competing interest.