Supplementary movies for the paper: Noriyuki Kodera, Daisuke Yamamoto, Ryoki Ishikawa, and Toshio Ando, "Video imaging of walking myosin V by high-speed atomic force microscopy", Nature (Article) 468: 72-76 (2010).

Supplementary Movie 1 (all-in-one QuickTime)

movie-1a, movie-1b, movie-1c, movie-1d (separate AVI files)

Supplementary Movie 1 | High-speed AFM movies showing unidirectional processive movement of M5-HMM. The dynamic process in 1 μM ATP was filmed at 146.7 ms frame^-1 (6.8 frames s^-1) and the movie is played at 7 frames s^-1. To track the M5-HMM molecules, the scan area was moved (a, c, d). Foot stomp events are marked with light-blue (L-head) and red triangles (T-head). (a) Scan range, 130 × 65 nm² with 80 × 40 pixels. Twelve frames are selected and shown in Fig. 1a. The time course of the molecular conformations is shown in the asterisk-labelled sequential boxes (Supplementary Fig. 3a). (b) Scan area, 125 × 62.5 nm² with 80 × 40 pixels. (c), (d) Typical movies showing long processive runs. (c) Scan range, 130 × 65 nm² with 80 × 40 pixels; the whole imaging area, 560 × 160 nm²; number of steps observed, 15. (d) Scan range, 150 × 75 nm² with 80 × 40 pixels; whole imaging area, 500 × 120 nm²; number of steps observed, 14.

　

Supplementary Movie 2  (all-in-one QuickTime)

movie-2a, movie-2b, movie-2c, movie-2d, movie-2e (separate AVI files)

Supplementary Movie 2 | High-speed AFM movies showing hand-over-hand movement of M5-HMM. The dynamic process was filmed at 146.7 ms frame^-1 (6.8 frames s^-1) and the obtained movies are played at 7 frames s^-1. Additional streptavidin molecules were scattered on the biotin-containing surface to slow the step movement. Foot stomp events observed at the L-head are marked with light-blue triangles (a, e). No foot stomping at the T-head is observed in these five movie clips. (a), (b) In 1 μM ATP. Scan area, 150 × 75 nm² with 80 × 40 pixels. (c) In 2 μM ATP. Scan area, 130 × 65 nm² with 80 × 40 pixels. Frames selected from (a) and (c) are shown in Figs. 1d and 1e, respectively. (d), (e) In 1 μM ATP. Scan area, 110 × 75 nm² with 80 × 50 pixels.

　

Supplementary Movie 3 (QuickTime)

movie-3 (AVI)

Supplementary Movie 3 | High-speed AFM movie of two-head bound M5-HMM observed in 50 μM ADP. The sample was filmed at 333.2 ms frame^-1 (3 frames s^-1) and the movie clip is played at 3 frames s^-1, from which 12 frames are selected and shown in Fig. 2a. The conformational states observed in the movie clip are shown in the first time course of Supplementary Fig. 3b (between the square brackets). Scan area, 90 × 90 nm² with 80 × 80 pixels. Foot stomp events observed at the L-head are marked with light-blue triangles. No foot stomping at the T-head is observed in this movie clip.

　

Supplementary Movie 4 (all-in-one QuickTime)

movie-4a, movie-4b, movie-4c, movie-4d (separate AVI files)

Supplementary Movie 4 | High-speed AFM movies showing unwinding of short coiled-coil tail of M5-HMM. (a), (b) Two different samples in 50 μM ADP were filmed at 333.2 ms frame^-1 (3 frames s^-1) and the obtained movies are played at 3 frames s^-1. AFM images selected from (b) are shown in Fig. 2b. Rewinding was not observed. Scan area, 90 × 90 nm² with 80 × 80 pixels. (c), (d) Two different samples in ADP were filmed at 333.2 ms frame^-1 (3 frames s^-1) and the obtained movies are played at 3 frames s^-1. Unwinding and rewinding of the tail were observed. Scan area, 90 × 90 nm² with 80 × 80 pixels. (c) In 50μM ADP, (d) in 1 mM ADP.

　

Supplementary Movie 5 (all-in-one QuickTime)

movie-5a, movie-5b, movie-5c (AVI) (separate AVI files)

Supplementary Movie 5 | High-speed AFM movies of two-head bound M5-HMM observed in the NF condition. Three different samples were filmed at 333.2 ms frame^-1 (3 frames s^-1) and the obtained movies are played at 3 frames s^-1. Foot stomp events at the L- and T-heads are marked with light-blue and red triangles, respectively. (a) Scan area, 90 × 90 nm² with 80 × 80 pixels. This movie corresponds to the AFM images shown in Fig. 2c and to the first time course shown in Supplementary Fig. 3c. (b) Scan area, 80 × 80 nm² with 100 × 100 pixels. This movie corresponds to the second time course shown in Supplementary Fog. 3c. (c) Scan area, 80 × 80 nm² with 100 × 100 pixels.

Supplementary Movie 6 (QuickTime)

movie-6 (AVI)

Supplementary Movie 6 | High-speed AFM movie showing transition between the straight and sharp bend conformations of the L-head. Actin-bound M5-HMM was imaged in the presence of 0.1 μM ADP at 3 frames s^-1 and the obtained movie is played at 12 frames s^-1. Scan area, 90 × 90 nm² with 80 × 80 pixels. The images are affected by the double-tip effect.