Fig. 3

Experimental design for high-speed observations of a myosin head in thick filaments. a A linker at position 3 in Fig. 1b is attached to a myosin head labeled with a 40 nm GNP. The other linkers are attached to the actin binding domains of α-actinin. b Schematic of a GNP attached to a myosin head. A handle sequence (red) projecting from the linker (dark blue) contains three handles bound to one antihandle labeled to myosin S1 (light blue) and two antihandles bound to GNP (orange). The head, oligonucleotides and GNP are drawn to scale. c An AFM image of the engineered thick filament anchored by α-actinin to an actin filament (red line) without myosin (all linkers are attached to α-actinin). The line profile is measured along the red and blue lines. The blue line was decided by shifting the red line in the perpendicular direction. The numbering of the linkers corresponds to Fig. 1b. Scale bar, 100 nm. d Line profiles along an actin filament (red line in c) and cross linkers (blue line in c). Measured values (dashed line) were fitted by multiple Gaussian distributions (solid line). The numbering corresponds to c. The distance axis indicates the relative distance from the position of the intensity peak (black dashed line) on the actin filament between linkers 3 and 4. e Averaged line profiles along the actin filament and cross linkers in d. Linkers 4, 5, and 6 are positioned between the peaks of an actin helical pitch. Linkers 1, 2, and 3 are not shown, because they frequently detached from actin in the absence of myosin. Shadowed areas indicate the standard error. n = 9. f A contour plot of a two-dimensional histogram of the wild-type S1 trajectory with a bin width of 5 nm. The elliptical shape is consistent with the prediction from the geometry of the complex (Supplementary Fig. 5). The two peaks of the plot correspond to the mean position of the detached state and the binding state, respectively.