Schematic of experimental setup for intermittent contact mode AFM.

Effects of feedback delay on the tip force exerted onto the sample. (a) Movement of Z-scanner (red line) tracing sinusoidally shaped sample surface (black line) with periodicity λ and amplitude h₀/2, when no parachuting occurs. Because of the delay of the Z-scanner movement, tracing error (blue line) is produced. (b) Tip force (blue line) exerted onto the sample when the set point force (black line) corresponding to the set point amplitude is relatively large. (c) Tip force (blue line) exerted onto the sample when the set point force (black line) is set at a small value.

Small cantilever. (a) Scanning electron microscopy (SEM) image of small cantilever. The inset shows an electron-beam-deposited (EBD) tip grown on an original bird-beak-shaped tip. (b) Magnified SEM image of EBD tip.

Amplitude and phase response curves of a cantilever as a function of excitation frequency when the cantilever is freely oscillating (solid line) and when the cantilever tip is influenced by negative force gradient (repulsive regime of tip–sample interaction) without energy dissipation (broken line). This interaction increases the cantilever’s resonant frequency. When the cantilever is excited at frequency f_ex, the resonant frequency shift results in amplitude reduction by ΔA and in phase advance by Δθ.

Streptavidin and its 2D crystal. (a) Schematic of streptavidin molecule placed on biotin lipid-containing lipid bilayer surface. ○, biotin-binding sites unoccupied with biotin; ●, biotin-binding sites occupied with biotin. (b) AFM image of streptavidin 2D crystal formed on biotin-containing SLB (top) and height profile along the white line shown in the AFM image (bottom).

Structure of myosin V, actomyosin ATPase reaction scheme, and prevailing view on conformational states of myosin. (a) Schematic of molecular structure of myosin V. (b) AFM image of myosin V. (c) Reaction scheme of actomyosin ATPase. (d) Schematic for backward leaning prestroke and forward leaning poststroke conformations. The mark “+” indicates the plus end of an actin filament (red). The motor domain (yellow) binds to an actin filament in the same orientation, in both conformational states, while the neck (blue) is leaned in different directions. The forward lever-arm swing is supposed to occur on the actin-bound head when Pi is released from myosin. The backward lever-arm swing is supposed to occur on the detached head when ATP is hydrolyzed to ADP–Pi.

Tail-truncated myosin V (M5-HMM) movement on actin filament captured by HS-AFM. All of the images were taken at a frame rate of 7 fps. (a) Successive AFM images showing processive movement of M5-HMM in 1 μM ATP when positively charged lipid is absent in the planar lipid bilayer (PLB) surface. The arrowhead indicates one of the streptavidin molecules attached to the PLB surface. (b) Successive AFM images showing processive movement of M5-HMM in 1 μM ATP when positively charged lipid is present in the PLB. The arrows indicate the coiled-coil tail pointing to the minus end of actin. The arrowhead indicates one of the streptavidin molecules attached to the PLB surface. (c) Clips of successive images showing long processive run of M5-HMM in 1 μM ATP (14 steps are recorded). (d) Schematic explaining structural features of two-headed bound M5-HMM observed in the presence of nucleotides. (e) Successive AFM images showing stepping process in 1 μM ATP. The swinging lever is highlighted with a thin white line.

Directional rule and its mechanism. (a) Typical HS-AFM images of two-headed M5-HMM showing the directional rule; there is a fixed relationship between the moving direction (arrows) and the direction (arrowheads) from which M5-HMM bind to an actin filament. (b) Schematic showing M5-HMM bound to actin at the left flanks of the motor domains. (c) Schematic showing the reason for the directional rule. In the M5-HMM shown in black, which does not follow the directional rule, the motor domains have to get into the narrow space between the actin filament and the substrate surface, which hardly occurs.

M5-HMM’s foot stomping at the leading head, unfolding of coiled-coil tail, and conformational transition of the leading head captured by HS-AFM. (a) Successive AFM images showing foot stomp at the leading head in the presence of 1 μM ATP. As pointed by the arrowheads, the leading head are briefly detached from actin. Because the detached head is moving fast, it does not appear in the images. Frame rate, 7 fps. (b) Unfolding of the coiled-coil tail of two-headed bound M5-HMM. Left images, before unfolding; right images, after unfolding. (c) HS-AFM images showing conformational transition between straight and bent conformations of the leading head in the presence of 0.1 μM ADP. The symbol “+” indicates the plus ends of actin filaments (b, c).

Displacement of motor domain of leading (a) and trailing (b) heads after foot stomping as a function of head separation before the foot stomping. Each right-hand figure shows the histogram of displacement.

Angle distributions of actin-bound single-headed M5. The single-headed M5 is formed by natural uncoiling of the short tail of two-head bound M5-HMM. (a) Schematic showing the angle measured. (b,d) HS-AFM images of bound single-headed M5 under the nucleotide-free condition (b) and in 1 mM ADP (d). The plus ends of actin filaments, which are judged from the directional rule shown in Figure , are indicated with “+”. The measured angles are shown in the images. Imaging rate, 3 fps. Scale bar, 30 nm. (c,e) Histograms of the angle measured for bound single-headed M5 under nucleotide-free condition (c) and in 1 mM ADP (e). The red curves represent the results of the best fit to the sum of two Gaussian distributions.

Leading head conformations and their dependence on nucleotides. (a) Population of straight and sharply bent conformations under different nucleotide conditions. The numbers in (frames) and [molecules] represent those of examined images and molecules, respectively. (b) Average lifetime ⟨τ_ST⟩ during which the leading head assumes the straight conformation in various ADP concentrations (the number of events used to obtain the average is shown in ⟨events⟩). The error bar indicates standard error. The line curve indicates the best fit result obtained by analyzing the data using the following equation: ⟨τ_st⟩ = n_NF/k_S–B + n_ADP/k_–, where k_S–B represents the spontaneous transition rate of the nucleotide-free leading head from the straight conformation to the sharply bent conformation, k_– represents the ADP dissociation rate constant for the leading head, and n_NF and n_ADP are, respectively, the fractions of nucleotide-free and ADP-bound leading head under a given ADP concentration, which can be calculated using the ADP dissociation constant K_d of 0.075 μM estimated from the data shown in (a).

HS-AFM imaging of α₃β₃ subcomplex of F₁-ATPase under different nucleotide states. (a) Crystal structure of α₃β₃γ subcomplex. The α-, β-, and γ-subunits are shown in cyan, pink, and yellow, respectively. (b) Typical HS-AFM image of α₃β₃ subcomplex immobilized on a chemically treated mica surface without nucleotide (150 × 150 pixels; imaging rate 1 fps). (c) Filtered HS-AFM image of C-terminal side of the α₃β₃ subcomplex without nucleotide. (d) C-terminal side of crystal structure of the nucleotide-free α₃β₃ subcomplex. The α and β subunits are colored in cyan and pink, respectively. The C-terminal DELSEED motif of the β subunits corresponding to the high protruding portions is highlighted in red. (e) Simulated AFM image constructed from the structure in (d). (f) Filtered AFM image of the N-terminal side of the α₃β₃ subcomplex without nucleotides. (g) N-terminal side of the crystal structure of the α₃β₃ subcomplex. (h) Simulated AFM image constructed from the structure in (g). (i) Filtered AFM image of the C-terminal side of the α₃β₃ subcomplex at 1 mM AMP-PNP. (j) Crystal structure of the α₃β₃ subcomplex with bound nucleotides. This structure is obtained by removing γ from the crystal structure of α₃β₃γ subcomplex. (k) Simulated AFM image constructed from the structure in (j).

HS-AFM imaging of α₃β₃ subcomplex in ATP. (a) Clips of successively captured HS-AFM images of the C-terminal side of α₃β₃ subcomplex in 2 μM ATP (imaging rate, 12.5 fps). The highest pixel in each image is indicated by the red circle. (b) Tight correlation between two types of conformational changes in β subunits observed in ATP; different heights of protrusion (“H”, high; “L”, low) and extended/retracted (“E”/”R”) conformations of the distal region. (c) Time course of cumulated angle of the highest pixel. The inset shows a trajectory, superimposed on an AFM image, of the highest pixels corresponding to the high protrusions of extended β subunits (412 frames). The center of rotation is defined by the averaged X- and Y-positions of the highest pixels, and the cumulated angles are calculated relative to the first frame.

Analysis of HS-AFM images of α₃β₃ subcomplex of F₁-ATPase using correlation coefficient. (a) Correlation coefficient histograms calculated for each β subunit (n = 220) observed in 2 μM ATP. An AFM image of an extended β subunit was used as a reference for the analysis of each β subunit. (b) Time courses of correlation coefficients. The white and gray backgrounds represent periods of extended high and retracted low states, respectively. Solid lines show the mean correlation coefficient for each period. (c) Time evolution of cumulated number of counterclockwise shifts of the RRE state. “○” and “×” correspond to RRE and other states (REE, EEE and RRR), respectively. The increase in the cumulated number indicates that the extended β subunit in RRE shifts counterclockwise. (d) Distributions of the number of successive shifts of the “RRE” state in clockwise (top) and counterclockwise (bottom) directions at 2 μM ATP. The values for “mean” indicate the average number of successive shifts, and the values for “s.d.” indicate the standard deviations.

Kinetics analysis of conformational change of α₃β₃ subcomplex. Histograms of dwell times for the E (top) and R (bottom) states in various concentrations of ATP (a, 2 μM, n = 266; b, 3 μM, n = 426; c, 4 μM, n = 374). Dwell time distributions for the E state fit well with single exponential decay functions (solid lines on top). The distributions for the R state fit with functions representing consecutive reactions with two identical time constants: A × t × exp(−t/τ) (solid lines on bottom). (d) ATP concentration dependence of the rate of conformational change of β subunit determined by fitting the dwell-time histograms of the E and R states, shown in (a)–(c), and the initial rate of ATP hydrolysis determined by biochemical assay at three different ATP concentrations.

Cessation of rotary conformational change propagation by loss of a single subunit. (a,d) AFM images before and after loss of (a) β₁ or (d) α₁ within α₃β₃ subcomplex in 2 μM ATP. Imaging rate, 12.5 fps. (b,e) Time courses of correlation coefficients for the three β subunits designated in (a) and (d), respectively. Solid horizontal lines show the mean correlation coefficients for the periods of E and R states. The states are judged by examining whether the correlation coefficients are above or below a threshold (0.978). β₁ was lost at 16.2 s (b) and α₁ was lost at 9.4 s (e) as indicated by the vertical broken lines. (c,f) Cumulated angles of open β subunit positions measured using the highest pixel position in each frame. (c) and (f) correspond to (b) and (e), respectively.

HS-AFM imaging of D96N bR mutant. (a,b) HS-AFM images of the cytoplasmic side (a) and the extracellular side (b) of D96N bR mutant (imaging rate, 1 fps) under dark (left panels) and green light illumination (right panels) (λ = 532 nm, 0.5 μW). bR trimers are highlighted by the white triangles. The insets in (a) display respective averaged images of trimers. The light-blue circles in (a) indicate a trefoil, and the green bars in (a) and (b) indicate green light illumination. (c) Decay after flash-illumination of the activated bR molecules observed at different pH values (7, 8, and 9). The exponential decay constants (τ) at pH 7, 8, and 9 are 6.7, 25, and 48 s, respectively. The inset shows the absorbance change at 410 nm (that is, decay of the M-intermediate) after flash illumination of D96N measured at various values of pH (7, 8, or 9). (d) Traces (red, blue, and purple marks) of the centers of mass of bR molecules under dark–illumination cycles are superimposed on the image of D96N in the dark. A bR trimer is encircled with the white triangle, and the positions of the α-helical ends facing the cytoplasmic surface (labeled with A–G) are derived from an atomic model of the unphotolyzed state.

Cooperative effects on the decay kinetics of D96N. (a) The top figure shows the decay of activated bR monomers (as depicted by the red circle), while the other monomers (the gray circles) within the respective trefoils are not in the activated state. The exponential decay constant is 7.3 ± 0.58 s. On the other hand, when one or two nearest neighbor molecules (as depicted by green circles in the middle figure) in a trefoil are already activated, the bR monomer activated latest (as depicted by the blue circles in the middle figure) decays faster. The exponential time constant is shortened to 2.0 ± 0.16 s. In contrast, the early activated molecules within a trefoil (as depicted by green circles in the bottom figure) show a nonexponential broad distribution of decay time with an average of 13.3 s. (b) Decay rate of activated D96N under different light intensities. The upper graph shows the decay of activated molecules under weak illumination. The decay constant is 5.4 ± 0.34 s. The lower graph shows the decay of activated molecules under relatively strong illumination. The decay constant is 6.1 ± 0.37 s.

Effect of alternate two-color illumination on the conformation of D96N bR observed by HS-AFM. (a) Displacement of centers of mass for three D96N bR molecules within a trimer measured as a function of time at pH 8 on the cytoplasmic side. The green bars show the periods of green light illumination. (b) Displacement of centers of mass for three D96N bR molecules within a trimer measured as a function of time at pH 8 on the cytoplasmic side. The green and blue bars show the periods of illumination of green and blue light, respectively. The average displacement induced by green light is 0.80 ± 0.13 (mean ± s.d.) nm. (c) Decay of the activated state of D96N bR at pH 8 detected by HS-AFM. Green and blue regions indicate application of green and blue light, respectively. Illumination periods of both green and blue light are 3 s.