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. 2018 Jul 20;7:e39775. doi: 10.7554/eLife.39775

Figure 1. Cryo-EM structures of SthK.

(A) Apo SthK side-view density map, colored by subunit. Dashed lines indicate the bilayer boundaries. The top is the extracellular side. Insets on the left are extracellular (top) and intracelluar (bottom) views. TMD-transmembrane domain, CNBD-cyclic nucleotide binding domain. (B) Atomic model of apo SthK colored as in (A). Insets on the right are extracelluar (top), and intracellular (bottom) views. (C) cAMP-bound SthK density map (grey) with bound cAMP highlighted in purple. (D) cGMP-bound SthK density map (grey) with bound cGMP highlighted in cyan. (E) Representative single-channel recording traces from SthK in horizontal lipid bilayers at +100 mV with 100 µM cAMP (top trace), 7 mM cGMP (middle), and no ligand (bottom). The zero-current level (closed channel) is indicated to the right by the letter C.

Figure 1.

Figure 1—figure supplement 1. Purification of SthK in lipid nanodiscs.

Figure 1—figure supplement 1.

(A) Size exclusion chromatography profile of SthK in lipid nanodiscs. (B) SDS-PAGE of SthK lipid nanodiscs showing both components (SthK monomer and MSP1E3). (C) Representative negative stain image with selected 2D classes (right) depicting the tetrameric nature of the channel in lipid nanodiscs. Scale bar is 100 nm and the box size is 330 Å.
Figure 1—figure supplement 2. Single particle cryo-EM of apo SthK.

Figure 1—figure supplement 2.

(A) Representative motion-corrected and dose-weighted micrograph of apo SthK in nanodiscs. (B) Selected 2D class averages. (C) Fourier shell correlation (FSC) curves between two independently refined half maps before (blue) and after (black) masking. Phase randomization was used to avoid effects from masking (red). (D) FSC curve from cross validation between the atomic model refined against half map 1 and half map 1 (blue), half map 2 (orange), and the summed map (grey). (E) Final map after Relion post-processing colored according to local resolution estimation using blocres. (F) Angular distribution of particles contributing to the final reconstitution. Scale bar is 50 nm and the box size is 281 Å.
Figure 1—figure supplement 3. Single particle cryo-EM of SthK in the precence of 2 mM cAMP.

Figure 1—figure supplement 3.

(A) Representative motion-corrected and dose-weighted micrograph of cAMP SthK in nanodiscs. (B) Selected 2D class averages. (C) Fourier shell correlation (FSC) curves between two independently refined half maps before (blue) and after (black) masking. Phase randomization was used to avoid effects from masking (red). (D) FSC curve from cross validation between the atomic model refined against half map 1 and half map 1 (blue), half map 2 (orange), and the summed map (grey). (E) Final map after Relion post-processing colored according to local resolution estimation using blocres. (F) Angular distribution of particles contributing to the final reconstitution. Scale bar is 50 nm and the box size is 275 Å.
Figure 1—figure supplement 4. Single particle cryo-EM of SthK in the precence of 7 mM cGMP.

Figure 1—figure supplement 4.

(A) Representative motion-corrected and dose-weighted micrograph of cGMP SthK in nanodiscs. (B) Selected 2D class averages. (C) Fourier shell correlation (FSC) curves between two independently refined half maps before (blue) and after (black) masking. Phase randomization was used to avoid effects from masking (red). (D) FSC curve from cross validation between the atomic model refined against half map 1 and half map 1 (blue), half map 2 (orange), and the summed map (grey). (E) Final map after Relion post-processing colored according to local resolution estimation using blocres. (F) Angular distribution of particles contributing to the final reconstitution. Scale bar is 50 nm and the box size is 281 Å.
Figure 1—figure supplement 5. Segmented density map of SthK-cAMP.

Figure 1—figure supplement 5.

Density map (grey mesh) determined from cAMP SthK shown together with the final atomic model (green). Densities shown are at a contour level of 5.5 σ.
Figure 1—figure supplement 6. Sequence alignment of members from the cyclic nucleotide modulated channel family.

Figure 1—figure supplement 6.

SthK (UniProtKB – G0GA88), human HCN1 (O60741), human HCN2 (Q9UL51), human CNGA1(P29973), human CNGA2(Q16280), TAX-4(Q03611), LliK(I0XVQ9), MloK1(Q98GN8). Secondary structure details labeled on top. Green: GYG signature sequence of potassium channel selectivity filter, cyan: charged residues in S4, light blue: counter charges, navy blue: charge transfer center, orange and yellow: conserved residues in C-linker and CNBD, respectively, grey: sequence not present and replaced by LESSGLVPRGSVKHHHH.