Expression and purification of CCK-8/CCK1R/G protein complexes.
(A, B) Snake plot of the CCK1R expression constructs for formation of mGsqi (A) or Gs (B) complexes. The construct for mGsqi complex formation contained an N-terminal HA signal sequence (grey shading), followed by a FLAG epitope tag (yellow shading), with a 3C protease cleavage site (red shading) inserted at the carboxyl terminus followed by an 8-His tag (green shading) and the mGsqi (blue schematic). For Gs complex formation, the construct contained an N-terminal HA signal sequence, followed by a FLAG epitope tag, a M4 mAChR N-terminal sequence (blue shading), and a 3C-cleavage site (red sequence) with an 8-His tag fused to the carboxyl terminus. (C) The expression constructs (red circles or blue circles) were cloned into a mammalian expression vector and concentration-responses to CCK-8 in an iCa2+ mobilisation assay were established relative to WT (black circles). (D, E) SEC of affinity purified CCK-8/CCK1R/mGαsqi/Gβ1γ2/scFv16 complex before (D) and after (E) sample cleavage with 3C protease; the peak used for SDS-PAGE analysis is boxed in red. (F) Coomassie blue stained SDS-PAGE of the peak sample from (D) (left panel) or (E) (right panel). (G) SEC of affinity purified CCK-8/CCK1R/DNGαs/Gβ1γ2/Nb35 complex. (H) Coomassie blue stained SDS-PAGE of the peak sample from (G). (I) amino acid sequence of the mGsqi chimera illustrating the origin of the different segments. (J, K) Gold standard FSC curves for the final map and map validation from half maps showing the overall nominal resolutions of 2.5 Å for the CCK1R-“Gq mimetic” complex (J) and 2.0 Å for the CCK1R–Gs complex (K). The data used to generate graph S1C, and the SEC traces in S1D, S1E, and SG are available in S1 Data. Uncropped gels for (F) and (H) are provided as S2 Data. Data for calculation of the FSC curves in S1J and S1K can be accessed in EMDB-23749 and EMDB-23750. CCK, cholecystokinin; CCK1R, cholecystokinin type 1 receptor; FSC, Fourier shell correlation; HA, hemagglutinin; SEC, size exclusion chromatography; WT, wild-type.
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