Figure 4: AFM analysis of MloK1 in the presence and absence of cAMP.

(a) High-resolution AFM topographs of MloK1. In the cAMP-bound state (left), each subunit of the tetrameric channels is well resolved on each single molecule. The four CNBDs were arranged in a left-handed windmill (inset: average). In the cAMP-free state (right), sub-molecular details could not be resolved and the tetramer is contoured as a single protrusion of variable height and appearance. (b) Height profile analysis of MloK1 molecules along the dashed lines in a. In the +cAMP conformation (left), the channels protruded 1.4±0.2 (mean±s.d.) nm (n=50) from the membrane. In absence of ligand (right), MloK1 protruded 2.9±0.4 nm (n=50). A lattice defect was exploited to ensure that the lower protrusion height of the +cAMP conformation was not underestimated owing to the dense molecule packing (height profile b1). (c) Time-lapse AFM of MloK1 channels undergoing the conformational change from cAMP-bound to -unbound state. MloK1 channels were imaged in the presence of 50 μM cAMP (t=0) after which the AFM fluid cell was extensively rinsed with cAMP-free buffer. After incubation time of 90 min, first single MloK1 channels lost their characteristic fourfold symmetric windmill structure and changed into a shapeless protrusion of increased height, and their number increased with time. The asterisks in the four image panels indicate the same membrane position.