Figure 5. Structure of HsTMEM120A at the CoASH-free state in comparison with the CoASH-bound state.

(A, B) The overall structure of HsTMEM120A without coenzyme A (CoASH) bound. The side view (A) and bottom view from intracellular side (B) are shown. The two monomers are colored light and dark green, respectively, while the intracellular loop IL5 is highlighted in orange. (C) Superposition of the structures of HsTMEM120A at the CoASH-free state (green for the bulk region and orange for IL5) and CoASH-bound state (silver). The view is similar to the one in the dashed box of panel (A). (D) Surface presentation of the region around the CoASH-binding site in the CoASH-free (left) and CoASH-bound (right) HsTMEM120A structures. The IL5 loop region is highlighted in orange. (E) Cryo-electron microscopy (cryo-EM) densities of the IL5 loop in the CoASH-free (left, contoured at 4.5 rmsd) and CoASH-bound (right, contoured at 3.3 rmsd) HsTMEM120A structures.

Figure 5—figure supplement 1. Cryo-EM data collection and processing of HsTMEM120A protein purified in detergent micelle.

(A) The flow chart of data collection, 2D classification, 3D classification, and refinement process. A total of 12,156 movie stacks were collected, and a representative motion-corrected micrograph is shown. Particles were subjected to several rounds of 2D and 3D classifications. The final map was generated by applying local refine procedure in cisTEM. (B) Angular distribution diagram of the particles contributing to the final reconstruction. (C) Estimation of the local resolution in the sharpened 4.0 Å cryo-electron microscopy (cryo-EM) map of HsTMEM120A protein. (D) The Fourier shell correlation (FSC) curves of the map with (red) or without (blue) an applied mask. The resolution was estimated according to the gold standard Fourier shell correlation (GSFSC) (FSC = 0.143) criterion. (E) Gel filtration profile ofHsTMEM120A protein eluted in a detergent solution. (F) Gel filtration profile of HsTMEM120A reconstituted in nanodiscs. Note the apparently lower A₂₆₀/A₂₈₀ peak ratio in (E) when compared to the one in (F). (G) The local area in the coenzyme A (CoASH)-binding cavity of the HsTMEM120A structure solved in the detergent. The elliptical dash ring indicates the CoASH-binding site as observed in the structure of the nanodisc complex. (H) A CoASH model from the nanodisc complex structure is superposed on the map region shown in (G). Note that there is no density feature accounting for the CoASH molecule in the cavity. The cryo-EM map at 4.0 Å shown in (G) and (H) is contoured at 5.0 V/4.5 rmsd level, while the other map at 4.2 Å resolution contoured at 0.25 V/3.5 rmsd level shows a similar weak density feature in the cavity and a more continuous density for the IL5 loop.

Figure 5—figure supplement 2. The occluded cavity of HsTMEM120A with CoASH bound in comparison with the open cavity without CoASH bound.

(A) Hole profiles of the coenzyme A (CoASH)-bound (left) and CoASH-free (right) HsTMEM120A structures. Red, pore radius < 1.15 Å; green, 1.15 Å < pore radius < 2.30 Å; blue, pore radius > 2.30 Å. The amino acid residues surrounding the narrowest site on the extracellular side and the CoASH molecule are shown as stick models in orange and pink, respectively. (B) Distribution of the pore radius along the central axes of the HsTMEM120A-CoASH complex and CoASH-free HsTMEM120A. The constricted area around CoASH on the intracellular side and four amino acid residues on the extracellular side are labeled on the right. (C) Cryo-electron microscopy (cryo-EM) densities of the four amino acid residues at the narrowest site on the extracellular side of HsTMEM120A at the CoASH-bound (left, contoured at 12.4 rmsd) and CoASH-free (right, contoured at 3.5 rmsd) states. (D) A mechanistic model accounting for two different functional states of TMEM120A in the cells.