Figure 2. Cryo-EM structure of inhibitor-bound TRPC4 channel.

(A) Side and top view of the cryo-EM map of GFB-8438 inhibitor-bound TRPC4, with each subunit colored differently. Positions of the transmembrane domain (TMD) and intracellular cytosolic domain (ICD) are indicated. (B) Side and top view of the structure of GFB-8438 inhibitor-bound TRPC4, with each subunit colored differently. (C) Location of non-protein densities relative to the atomic model of TRPC4, which is shown in transparent ribbon representation in the side- and top view. Densities corresponding to lipids are depicted in red, GFB-8438 density is shown in purple. (D) Close-up of the ligand-binding pocket, showing the density corresponding to the inhibitor GFB-8438(transparent) with the ligand structure modelled inside. GFB-8438 is enclosed by the four helices S1 to S4, constituting the VSL domain. A rotated view of the ligand-binding pocket is shown in the left panel with important and interacting residues highlighted. GFB-8438 is shown in purple. In the right panel the chemical structure of the TRPC4 inhibitor GFB-8438 is shown, with important and interacting residues of TRPC4 highlighted. Non-carbon atoms are colored according to element, with halogens in green, nitrogen in blue and oxygen in red. (E) and (F) Same in (D) but for inhibitor GFB-9289 and GFB-8749 bound structures of TRPC4 respectively.

Figure 2—figure supplement 1. Cryo-EM image processing workflow for TRPC4 in complex with the inhibitor.

(A) The top panel shows a representative digital micrograph area and selected 2-D class averages of inhibitor GFB-8438 bound TRPC4. Scale bars, 50 nm and 10 nm, respectively. The initial refinement density and subsequent densities obtained after 3D classification are shown next to the 2-D class averages and in the bottom panel, respectively. (B) Angular distribution of particles used in the final refinement and Fourier shell correlation curves (FSC) between the two independently refined maps. The dotted lines indicate the 0.143 FSC criterion used for average resolution estimation. (C, D) Same as in (A) and (B), respectively, but for the inhibitor GFB 9289-TRPC4 complex. (E, F) Similar to (A) and (B), respectively representing inhibitor GFB-8749-TRPC4 complex.

Figure 2—figure supplement 2. Cryo-EM map of TRPC4 in complex with the inhibitor GFB-9289 and GFB-8749.

(A) Side and top view of the cryo-EM map of GFB-9289 inhibitor-bound TRPC4, with each subunit colored differently. Positions of the transmembrane domain (TMD) and intracellular cytosolic domain (ICD) are indicated. (B) Location of non-protein densities relative to the atomic model of TRPC4, which is shown in transparent ribbon representation in the side- and top view. Densities corresponding to lipids are depicted in red, GFB-9289 density is shown in blue. (C) Same as in (A), but for the cryo-EM map of GFB-8749 inhibitor-bound TRPC4. (D) Same as in (B), except the density for GFB-8749 is shown in green.

Figure 2—figure supplement 3. Local resolution of the TRPC4-ligand complex maps.

Maps of GFB-8438, GFB-9289 and GFB-8749-bound TRPC4, respectively, colored according to the local resolution. Representative regions of the density with the fitted atomic model are shown below the local resolution maps.

Figure 2—figure supplement 4. Comparison between different TRPC4 structures.

(A) Structural alignment of a protomer of the inhibitor GFB-8438 bound TRPC4 with that of TRPC4 in its apo state. The protomer of the TRPC4 apo structure is shown in cartoon representation and colored in light blue while the protomer of the inhibitor bound TRPC4 structure is colored in light red. (B) Same as in (A) for the inhibitor GFB-9289 bound TRPC4. The protomer TRPC4 structure is shown in light green. (C) Similar to (A), but for inhibitor GFB-8749 bound TRPC4 structure colored in cyan. (D) Alignment of the structures of TRPC4 solubilized in amphipols or in the detergent LMNG. The protomer of apo-LMNG structure is shown in dark blue. (E) Alignment of the TRPC4-CaM complex structure with the TRPC4 apo-LMNG structure. The TRPC4-CaM complex is also solubilized in LMNG. The protomer of the TRPC4-CaM structure is shown in purple. (F) Alignment of the TRPC4-inhibitors and TRPC4-CaM structures. Note: The C-terminal helix in the apo structure (PDB ID: 6GIK) has been corrected for domain swapping (See Method section and Figure 5—figure supplement 3B for details).

Figure 2—figure supplement 5. Domain architecture of zebrafish TRPC4 channel.

Cartoon representation of a TRPC4 protomer. Each domain is shown in a different color and labeled accordingly.