(A) Side-by-side comparison of cGMP-bound SthK CNBD from cryo-EM (green) and X-ray crystallography for the isolated domain (wheat). The green is in the resting and the wheat is in the activated conformation as shown by the C-helix position. cGMP configuration is in syn (green) and anti (wheat). (B) Overlays between apo SthK (green), apo HCN1 (yellow), and cAMP HCN1 (blue) CNBDs (left) and siphon regions (right). The C-helices are visible in lower, intermediate and upper position, respectively and the siphon loops are visible correspondingly in a lower, intermediate, and upper position. (C) Overlay between CNBDs of cyclic nucleotide-modulated ion channels shows two major conformational states of the C-helix (depicted as a cylinder), either in lower (resting) conformation or upper (activated) conformation. C-helix of apo HCN1 (yellow) is in an intermediate conformation, but closer to the upper (activated) conformation. (E) Overlays between siphon regions of apo SthK (green, left) and apo HCN2 (purple), showing close to identical conformations. The overlays between cryo-EM SthK (green; cAMP (middle) and cGMP (right)) and the crystal structure SthK (grey and wheat, cAMP and cGMP, respectively) siphons show big differences, corresponding to the large differences between resting and activated CNBDs. PDB codes: apo SthKEM (6CJQ), cAMP SthKEM (6CJU), cGMP SthKEM (6CJT), cAMP SthKXRAY (4D7T), cGMP-SthKXRAY (4D7S), apo HCN1 (5U6O), cAMP HCN1 (5U6P), TAX-4 (5H3O), LliK (5V4S), apo HCN2 (5JON), cAMP HCN2 (1Q43), cGMP HCN2 (1Q3E), MolK1 (6EO1).