Figure 5. Structure of the human CLC-7/OSTM1 complex.
(A–B) Cryo-EM density map (A) and structure (B) of CLC-7/OSTM1 complex viewed from within the membrane (left), the cytosol (middle) and the lysosomal lumen (right). CLC-7 is colored by domain with N-terminal domain in magenta, transmembrane domain in blue, CBS1 in cyan and CBS2 in green. OSTM1 is colored yellow. Modeled non-protein densities are colored orange and unmodeled non-protein densities are colored grey in A.
Figure 5—figure supplement 1. Cryo-EM analysis of human CLC-7/OSTM1.
(A) Representative cryo-EM image of hsCLC-7/OSTM1. (B) Two-dimensional class averages. (C) Simplified image processing workflow. (D) Fourier shell correlation (FSC) of two unfiltered half-maps for consensus (black), TMD focus refine (red), CD focus refine (green) and LD focus refine (orange) reconstructions. (E) Cross-correlation plot of two unfiltered half-maps following density modification for consensus (black), TMD focus refine (red), CD focus refine (green) and LD focus refine (orange) reconstructions. (F) FSC of map-to-model fit of human CLC-7/OSTM1 with composite density modified map. (G) Angular distribution. (H) ThreeDFSC anisotropy plots.
Figure 5—figure supplement 2. Three-dimensional variability analysis of human CLC-7/OSTM1.
Cryo-EM density maps (A) and models (B) revealing three orientations of the OSTM1 luminal domain. The red map is the origin (frame 000) of the second major component of 3DVA, the yellow map represents the midpoint (frame 010) and the blue map represents the end point (frame 019). The largest displacement occurs in helix h6, which moves by ~6 Å between the two extreme states. CLC-7 is colored grey in B.
Figure 5—figure supplement 3. Representative cryo-EM density and model of human CLC-7/OSTM1.
(A) Representative sections of cryo-EM density shown as grey mesh displayed at 8 σ threshold. Refined coordinates are shown as sticks. (B) Density for modeled non-protein ligands. (C) Unmodeled density for potential lipid-binding site at the interface between CLC-7 and OSTM1. CLC-7 is colored by domain with, transmembrane domain in blue. OSTM1 is colored yellow.
Figure 5—figure supplement 4. Comparison of ligand-binding sites in ggCLC-7 and human CLC-7/OSTM1.
(A) ATP-binding sites in ggCLC-7 (colored in grey) and hsCLC-7/OSTM1 (colored by domain). Black numbering is ggCLC-7 and colored numbering is hsCLC-7/OSTM1. (B) PI3P binding sites in ggCLC-7 (colored in grey) and hsCLC-7/OSTM1 (colored by domain). Black numbering is ggCLC-7 and colored numbering is hsCLC-7/OSTM1.