Cryo-EM structure of the endothelin-1-ETB-Gi complex

  1. Fumiya K Sano
  2. Hiroaki Akasaka
  3. Wataru Shihoya  Is a corresponding author
  4. Osamu Nureki  Is a corresponding author
  1. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan
3 figures, 2 tables and 1 additional file


Figure 1 with 2 supplements
Overall structure of the ET-1-ETB-Gi signaling complex.

(A) Schematic representations of the fusion-G system. (B) Fluorescence-detection size-exclusion chromatography (FSEC) analysis of complex formation by the ETB receptor. The fluorescence intensities are adjusted to equalize those corresponding to the void volumes. (C) Cryo-electron microscopy (cryo-EM) map with variously colored densities. (D) Structure of the complex determined after refinement in the cryo-EM map, shown as a ribbon representation. (E) Density focused on ET-1.

Figure 1—figure supplement 1
Fusion-G system.

(A, B) Fluorescence-detection size-exclusion chromatography (FSEC) analysis of the complex formation of LPA1 (A) and S1P5 (B). The fluorescence intensities are adjusted to equalize those corresponding to the void volumes. When co-expressed with the Gi trimer, the peak corresponding to the complex appears on the high molecular weight side of the receptor, reflecting the basal activity and the HiBiT-LgBiT binding, independent of the G-protein. When the co-expressed cells were treated with the agonists and apyrase, the complex peak of LPA1 and S1P5 became larger, corresponding to the GPCR-G-protein complex. (C) Size-exclusion chromatography elution profiles of the ETB -Gi complex. (D) SDS-PAGE analysis of the gel filtration fractions. Although the unbound receptor was present, the complex could be separated and purified effectively.

Figure 1—figure supplement 2
Cryo-electron microscopy (Cryo-EM) workflow, maps, and model quality.

Flow chart of the cryo-EM data processing for the ETB -Gi complex, including particle projection selection, classification, and three-dimensional (3D) density map reconstruction. Local resolution maps, FSC curves, and cryo-EM density maps are also shown. Unless otherwise noted, analysis jobs were run on cryoSPARC v3.3. Details are provided in the Methods section.

Structural changes upon G-protein activation.

(A) Superimposition of the ET-1-bound receptor in the crystal and cryo-electron microscopy (cryo-EM) structures. (B) Superimposition of the ETB structures, focused on the receptor core. (C, D) Superimpositions of the Gi-complexed ETB structure with the ET-1-bound crystal structure (C) and apo structure (D). (E-G) D3.49R3.50Y3.51 and N7.49P7.50xxY7.53 motifs in ETB (E), β2AR (F), and μOR (G). Black dashed lines indicate hydrogen bonds.

Figure 3 with 2 supplements
Comparison of the Gi binding modes.

(A) Hydrogen-bonding interactions between ETB and the α5-helix, indicated by black dashed lines. (B) Comparison of the Gα positions in the GPCR-G-protein complexes. The structures are superimposed on the receptor structure of the NTS1-C state.

Figure 3—figure supplement 1
Detailed ETB-Gi interface.

(A, B) Receptor-Gi interactions within 4.5 Å. Black dashed lines indicate hydrogen bonds. (C-E) Structural comparisons of the interactions between ICL2 and Gi in ETB (C), S1P1 (D), and CB1 (E). Residues are shown as stick and CPK models. (F) Structural comparison of the Gαi1 subunits.

Figure 3—figure supplement 2
Comparison of the interactions between the α5-helix and TM7-H8.

(A-F) Interactions between the α5-helix and TM7-H8 within 4.5 Å in the respective complexes.


Table 1
Cryo-EM data collection, refinement, and validation statistics.
Data collectionETB-Gi (overall)ETB-Gi (receptor focused)
MicroscopeTitan Krios (Thermo Fisher Scientific)
Voltage (keV)300
Electron exposure (e-/Å2)49.965
DetectorGatan K3 summit camera (Gatan)
Defocus range (μm)–0.8–1.6
Pixel size (Å/pix)0.83
Number of movies10,408
Picked particles3,863,134
Final particles260,085
Map resolution (Å)2.803.13
FSC threshold0.143
Model refinement
R.m.s. deviations for ideal
Bond lengths (Å)0.0020.003
Bond angles (°)0.560.52
Rotamers (%)0.000.00
Ramachandran plot
Favored (%)96.5598.04
Allowed (%)3.191.96
Outlier (%)0.260.00
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
peptide, recombinant proteinET-1PEPTIDE INSTITUTE, INC.Cat #,
4198 v
Ligand for ETB
OtherSf-900 II SFMThermo Fisher ScientificCat #,
Expression medium for sf9 cells
chemical compound, drugn-dodecyl-β-D-maltosideCalbiochemCAS number:
Detergents used in purification of ETB-Gi complex
chemical compound, drugCholesteryl hemisuccinateMerck MilliporeCAS number:
For purifying ETB-Gi complex
peptide, recombinant proteinApyraseNew England BiolabsCat #,
Enzyme used for ETB-Gi complex formation
OtherAnti-DYKDDDDK G1 Affinity resinGen ScriptCat #,
Affinity resin for DYKDDDDK tags
chemical compound, drugLauryl Maltose Neopentyl GlycolAnatraceCAS number:
Detergents used in purification of ETB-Gi complex
Software, algorithmEPUThermo Fisher ScientificFor Cryo-EM data collection
Software, algorithmRELION-3.1Zivanov et al., 2018RRID:SCR_016274For Cryo-EM data processing
Software, algorithmcryoSPARC v3.3STRUCTURA BIOTECHNOLOGYRRID:SCR_016501For cryo-EM data processing
Software, algorithmCootEmsley et al., 2010RRID:SCR_014222For structure model building
Software, algorithmPhenix 1.19–4092Afonine et al., 2018RRID:SCR_014224For structure refinement
otherQuantifoil holey carbon gridQuantifoilR1.2/1.3, Au, 300 meshFor cryo-EM specimen preparation

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  1. Fumiya K Sano
  2. Hiroaki Akasaka
  3. Wataru Shihoya
  4. Osamu Nureki
Cryo-EM structure of the endothelin-1-ETB-Gi complex
eLife 12:e85821.