Supplementary information forVideo imaging of walking myosin V by high-speed atomic
force microscopy
Noriyuki Kodera1,2, Daisuke Yamamoto1,2, Ryoki Ishikawa3, Toshio Ando1,2*
1Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan. 2CREST, JST, Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. 3Department of
Molecular and Cellular Pharmacology, Gunma University Graduate School of
Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
*Correspondence and request for materials should be addressed to T.A.
(E-mail: tando@kenroku.kanazawa-u.ac.jp)

This document contains:
1. One Supplementary Table with legend
2. Six Supplementary Figures with legends
3. Six legends for Supplementary Movies

1Supplementary Table 1 | Summary of kinetic parameters. Kinetic parameters wereobtainedusing
therelationships
V = Vmax / (1 + K m / [ATP ])andV = d s / (1 / k1 [ATP ] + 1 / k 2 ) , where V is the average translocation velocity in a given[ATP] (Supplementary Fig. 2), Vmax is the maximum translocation velocity, Km is theapparent Michaelis-Menten constant, ds is the average step size, and k1 and k2 are thesecond-order ATP binding rate constant and first-order ADP dissociation rate constant,respectively. The second relationship holds because ADP release is a major rate-limiting
step, ATP binding is also a rate-limiting step in low [ATP], other chemical steps occur
much faster1, and one discrete translocation is tightly coupled to one ATP hydrolysis2.
All values represent best-fit results ± s.d. When DPTAP-free lipid bilayers were used,
Vmax was 403 ± 7 nm s-1, which is similar to that obtained using fluorescencemicroscopy under the same buffer condition3,4, indicating that tip-sample andsurface-sample interactions have negligible effects on motor activity. However, in theabsence of surface-sample attraction, M5-HMM occasionally did not travel sideways
along actin filaments; hence, it was difficult to acquire high-resolution images since
AFM is inefficient for visualising molecules not supported on a surface. Therefore, theaddition of the positively charged lipid was necessary to improve image quality andexperimental efficiency, despite a modest decrease in average translocation velocity.

2Supplementary Figure 1 | Schematic of assay system for HS-AFM imaging (notscaled). A mica surface was fully covered with biotin-containing lipid bilayers.
Streptavidin molecules (green circles) were partially deposited on the substrate.
Biotinylated actin filaments were immobilised on the bilayer surface throughstreptavidin molecules. M5-HMM was deposited on the lipid bilayers. All imaging
experiments were performed in the tapping mode using a laboratory-built high-speed
AFM apparatus5,6.

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Supplementary Figure 2 | Translocation velocity of M5-HMM observed by
HS-AFM. The observations were performed in various [ATP] using planar lipid bilayer
surfaces containing different fractions of positively charged lipid DPTAP. Walking
M5-HMM molecules were imaged at 146.3 ms frame-1 (400 × 125 nm2 scan range, 80 ×25 pixels). In ATP higher than 20 μM, very shortly dwelled states were missed. In thiscase, translocation velocity was obtained from movies in which at least four instances ofdwelling at different locations along an actin filament were captured on video. The
translocation velocity in a given [ATP] was thus measured and its average (V) was fitted
to the relationship V = d s / (1 / k1 [ATP ] + 1 / k 2 ) (see Supplementary Table 1). The
curves are best-fit results. Each data point was obtained from the translocation
trajectories of more than 100 molecules (103-276). Error bars, ± s.d. The fitting results
are summarised in Supplementary Table 1.

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Supplementary Figure 3 | Time courses showing the molecular conformations
under different nucleotide conditions. (a) Six typical time courses in 1 μM ATP. Eachtime course starts from the right-pointing triangle and ends with the left-pointing

5triangle. Discrete ~36 nm steps occur at the vertical dashed lines. Each box corresponds
to a video frame. The colour code of the boxes is given at the bottom of the figure. The
same colour code is also used for (b) and (c). Each box with a black dot, which iscontained in the asterisk-labelled time course, corresponds to an AFM image shown in
Fig. 1a (Supplementary Movie 1a). Scale bar, 1 s. (b) Two typical time courses in 50
μM ADP. Each box with a black dot in the first time course corresponds to an AFM
image in Fig. 2a (Supplementary Movie 3). Scale bar, 3 s. (c) Three typical time courses
in the NF condition. Each box with a black dot in the first time course corresponds to an
AFM image in Fig. 2c (Supplementary Movie 5a). The molecule observed in the second
time course is shown in Supplementary Movie 5b. Scale bar, 3 s.

6Supplementary Figure 4 | Angle distributions of single-headed M5 bound to actin.
The single-headed M5 is formed by natural unwinding of the short tail of two-headbound M5-HMM. (a) Schematic showing the angle measured. (b), (d) AFM images ofsingle-headed bound M5 in the NF and 1 mM ADP conditions, respectively. The plus
ends of actin filaments are indicated with ‘+’. The measured angles are shown in theimages. Imaging rate, 333.2 ms frame-1. Scale bar, 30 nm. (c), (e) Histograms of theangle measured for single-headed bound M5 in the NF and 1 mM ADP conditions,
respectively. Blue broken lines, single-Gaussian fit results; black solid lines,
double-Gaussian fit results; red lines, Gaussian components in double-Gaussian fit. Allfits were restricted to bins with > 10 counts. All values for peak angles represent mean ±s.d. The two-Gaussian distributions are statistically significant judging from the F-test
results: p < 0.05 for the NF condition; p < 10-6 for the 1 mM ADP condition.

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Supplementary Figure 5 | Proportion of foot stomp events that occur at L- andT-heads in various [ADP]. The proportion depends on the L-head conformation(straight or sharp bend); the proportion difference between the L- and T-heads becomes
smaller when the L-head assumes the sharp bend conformation, irrespective of [ADP].
In 50 μM ADP, the L-head almost always assumes the straight conformation. Foot
stomp events counted without distinguishing between the two conformations of theL-head are indicated by ‘all’. The numbers in <events>, [molecules], and (frames)
represent the respective numbers examined for foot stomp events observed under therespective conditions.

8Supplementary Figure 6 | Walking mechanism of myosin V highlighting the energy
landscapes of actin-bound L- and T-heads. The red lines represent the energy
landscapes of actin-bound heads in the respective states. The blue lines represent theenergy landscapes of detached heads. The left and right sides partitioned by the verticaldashed lines of each energy landscape shown in red correspond to the reversearrowhead (R-ARH) and arrowhead (ARH) conformations, respectively. The left and9right sides partitioned by the vertical lines of each energy landscape shown in blue
correspond to the pre-stroke and post-stroke conformations, respectively. The black dot
in each energy landscape indicates the most probable position of the corresponding headin the energy landscape. Each energy landscape shown in a faint colour represents thatin the previous state. (a) Initial state of the step cycle in which both heads (H1 and H2)carry bound ADP. In this chemical state, foot stomping more frequently occurs at theL-head than at the T-head, and therefore, the free energy level of the L-head is higher
than that of the T-head. The L-head-bound ADP is very stable. (a’) State in which theL-head detaches from actin while foot stomping. The detached L-head is in equilibrium
between the pre-stroke and post-stroke conformations. The T-head is in thelowest-energy state as the intramolecular strain is released. Even in this state, the T-headcan swing backward with a low probability. Transitions between (a) and (a’) occur
alternately. (b) ADP release, subsequent ATP binding, and resulting detachment at the
T-head. The T-head detachment removes the high potential barrier in the energy
landscape of the L-head, thus, the L-head spontaneously descends to the bottom of theenergy landscape, swinging from the R-ARH orientation to the ARH orientation. The
T-head rotationally diffuses around the advancing neck-neck junction to search for an
actin subunit to attach to. The ADP-Pi-bound L-head possibly preferentially assumes the
pre-stroke conformation as reported previously7,8. (c) The detached head is reattachedand then phosphate is immediately released to return to the original two-head boundstate (a), resulting in a ~36 nm advance. As the sharp bend conformation is rarely
observed on the leading neck even in low [ATP] (0.1 μM and 1 μM), M5 only moves
forward by repeating the changes described in (a), (b), and (c) in this order. (d) The
sharp bend conformation is frequently observed in the leading neck in the NF condition.
In this structural state, the two heads are bound to actin in the ARH orientation (at least
around the motor domains) with similar affinities, and thus, the foot stomp occurs on thetwo heads at nearly equal frequencies.

10Legends for Supplementary Movies
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 with80 × 40 pixels. Twelve frames are selected and shown in Fig. 1a. The time course of themolecular 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 | High-speed AFM movies showing hand-over-handmovement 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 | 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) andthe 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 timecourse 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 withlight-blue triangles. No foot stomping at the T-head is observed in this movie clip.
Supplementary Movie 4 | High-speed AFM movies showing unwinding of shortcoiled-coil tail of M5-HMM. (a), (b) Two different samples in 50 μM ADP were filmed11

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. Scanarea, 90 × 90 nm2 with 80 × 80 pixels. (c), (d) Two different samples in ADP werefilmed at 333.2 ms frame-1 (3 frames s-1) and the obtained movies are played at 3 framess-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 | 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) Scanarea, 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) Scanarea, 80 × 80 nm2 with 100 × 100 pixels. This movie corresponds to the second timecourse shown in Supplementary Fig. 3c. (c) Scan area, 100 × 100 nm2 with 80 × 80
pixels.
Supplementary Movie 6 | High-speed AFM movie showing transition between thestraight and sharp bend conformations of the L-head. Actin-bound M5-HMM wasimaged 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.

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