Extended Data Fig. 2: Full-length GPR56 purification, cryo-EM processing and low resolution 3D maps for NTF bound GPR56 and LPHN3.
From: The tethered peptide activation mechanism of adhesion GPCRs
a, Design for the full-length cleavage deficient (CD, H381S) GPR56 construct used for structural studies. Human GPR56 (V2-FL) was cloned into pFastBac containing a hemagglutinin signal peptide tag (HA) and a cleavable N-terminal FLAG-tag. The construct included a C-terminal cleavable His6 tag. b, Size exclusion chromatography (SEC) profile of full-length GPR56. Fractions corresponding to the monomeric peak (grey) were collected and used for structural studies. c, Coomassie stained SDS-PAGE of the FL-GPR56 sample used for cryo-EM analysis. d, Cryo-EM data processing workflow of the FL-GPR56 sample. e, f. Low resolution 3D maps of NTF-bound receptor conformations with docked structures of the ECR of e, GPR56 and f, LPHN3. The GAIN domains and TA peptides are colored in blue and cyan for GPR56 and in magenta and light pink for LPHN3. Domains are labeled. Docked available ECR crystal structures corresponding to PDB 5KVM and 4DLQ for GPR56 and LPHN1, respectively. Scale bars are provided in the left bottom corner.
