www.sciencemag.org/cgi/content/full/333/6043/755/DC1

Supporting Online Material for
High-Speed Atomic Force Microscopy Reveals Rotary Catalysis of
Rotorless F1-ATPase
Takayuki Uchihashi, Ryota Iino, Toshio Ando,* Hiroyuki Noji**To whom correspondence should be addressed. E-mail: hnoji@appchem.t.u-tokyo.ac.jp (H.N.);
tando@staff.kanazawa-u.ac.jp (T.A.)
Published 5 August 2011, Science 333, 755 (2011)
DOI: 10.1126/science.1205510
This PDF file includes:
Materials and Methods
Figs. S1 to S12
Table S1
References (26–28)
Other Supporting Online Material for this manuscript includes the following:(available at www.sciencemag.org/cgi/content/full/333/6043/755/DC1)
Movies S1 to S8

Supporting Online Materials

High-speed atomic force microscopy reveals rotary catalysis ofrotor-less F1-ATPase
Takayuki Uchihashi, Ryota Iino, Toshio Ando, Hiroyuki Noji
MATERIALS AND METHODS
Purification of α3β3 subcomplex and measurement of ATP hydrolysis rate
Fixation of α3β3 subcomplex onto substrate for AFM observation
High-speed AFM apparatus
AFM imaging and image processing
Correlation analysis
Measurement of cumulated number of CCO shifts
Measurement of cumulated rotational angle
Calculation of the degree of underestimation of unidirectionality
Simulation of AFM image
SUPPORTING FIGURES S1 TO S12
S1: Purification and ATPase activity of α3β3 subcomplex
S2: Comparison of raw, temporally filtered, and spatially filtered AFM images
S3: Wide-area AFM images of α3β3 subcomplex without nucleotide and in 2 mM ATP
S4: Three dimensional illustrations of the crystal structure and AFM image innucleotide-free condition.
S5: Experimental and simulated AFM images of the N-terminal side of α3β3
subcomplex
S6: Tight correlation between two types of conformational changes in β observed inATP
S7: Reference AFM images and ROIs used in the correlation analysis of βconformations observed in ATP
S8: Correlation coefficient analysis of three βs in α3β3 subcomplex observed innucleotide-free condition
S9: Correlation coefficient analysis of three βs in α3β3 subcomplex observed in
AMPPNP
S10: Distributions of the number of successive shifts of “CCO” state in clockwise andcounterclockwise directions
1S11: Histograms of dwell times for the O and C states in various [ATP]S12: Loss of a single subunit stops rotary propagation of conformational changes
SUPPORTING TABLE S1
S1: Fraction of molecules showing conformational changes in various nucleotide
conditions.
SUPPORTING ONLINE MOVIES S1 TO S8
Movie S1: AFM movie of the C-terminal side of α3β3 without nucleotide. Scan area, 18
× 15 nm2; frame rate, 10 fps.
Movie S2: AFM movie of the N-terminal side of α3β3 without nucleotide. Scan area, 17
× 11 nm2; frame rate, 10 fps.
Movie S3: AFM movie of the C-terminal side of α3β3 in 2 μM ATP. Scan area, 17 × 13
nm2; frame rate, 12.5 fps.
Movie S4: AFM movie of the C-terminal side of α3β3 in 3 μM ATP. Scan area, 18 × 14
nm2; frame rate, 12.5 fps.
Movie S5: AFM movie of the C-terminal side of α3β3 in 4 μM ATP. Scan area, 17 × 11
nm2; frame rate, 12.5 fps.
Movie S6: AFM movie of the C-terminal side of α3β3 in 2 μM ATP. Scan area, 21 × 14
nm2; frame rate, 12.5 fps. The pixel with the highest (brightest) position ineach image is indicated by the blue circle. The center used for calculating
the rotational angle is indicated by the cross mark.
Movie S7: AFM movie of the C-terminal side of α3β3 in 2 μM ATP. Scan area, 21 × 14
nm2; frame rate, 12.5 fps. At 16 s, one β subunit was lost.
Movie S8: AFM movie of the C-terminal side of α3β3 in 2 μM ATP. Scan area, 25 × 14
nm2; frame rate, 12.5 fps. At 9 s, one α subunit was lost.

2

MATERIALS AND METHODS
Purification of α3β3 subcomplex and measurement of ATP hydrolysis rate
The α(His6 at N-terminus/C193S)3β(His3-Lys7 at N-terminus)3 subcomplex of the
F1-ATPase from thermophilic Bacillus sp. PS3 was expressed in E. coli, and purified
using Ni2+-NTA affinity chromatography and size exclusion chromatography as
described previously for purification of the α3β3γ subcomplex (26) (Fig. S1). The
nucleotide-free α3β3 subcomplex was stable and stored at room temperature before use.
The ATP hydrolysis rate was measured with an ATP regenerating system using a
UV-visible spectrophotometer (VP-550, Jasco). Various concentrations of ATP wereadded to the assay mixture (10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 250 mM KCl, 2.5mM phosphoenolpyruvate, 0.1 mg/ml pyruvate kinase, 0.1 mg/ml lactate dehydrogenase,and 0.2 mM NADH) at 0 s, followed by the addition of α3β3 (final 30 nM) at 200 s. The
NADH absorbance at 340 nm was monitored and the initial rate of ATP hydrolysis wascalculated from the initial slope (for 10 s after the addition of α3β3) of the change in[NADH] using a molecular extinction coefficient of 6,220. Measurements were carriedout at 24–25 °C (Fig. S1).
Fixation of α3β3 subcomplex onto substrate for AFM observation
To fix the α3β3 subcomplex onto a mica substrate, we first treated the mica surface with
3-aminopropyl-triethoxysilane (0.05–0.1%) for 3 min and washed the surface with purewater. The mica was then treated with glutaraldehyde (0.1–0.25%) for 3 min andcarefully washed with buffer A (10 mM HEPES-NaOH (pH 7.4), 10 mM KCl, 5 mM
MgSO4). A droplet containing the α3β3 subcomplex (1–10 nM) was deposited on thesurface for 5 min, which was then washed with buffer B (10 mM Tris-HCl (pH 8.0), 2
mM MgCl2). The sample stage was mounted on the AFM head and immersed in buffer
B with or without ATP or AMPPNP. AFM imaging showed that the α3β3 subcomplex
was strongly adsorbed on the mica surface by chemical cross-linking between the
primary amine on the aminosilane-modified mica and the ε-amines of the lysine
residues in the N-terminus of the β subunit. Judging from the observed images andheight of the molecules (~9 nm), almost all molecules appeared to be adsorbed on themica at either the C- or N-terminal side. Most (~70%) of the adsorbed molecules had
the C-terminal side facing upward, whereas the rest had the N-terminal side facing
upward. On bare and untreated mica surfaces, all molecules rapidly diffused on thesurface and could not be imaged clearly.

3

High-speed AFM apparatus
We used a home-built high-speed AFM apparatus (17, 18). The cantilevers (Olympus)
were 6–7 μm long, 2 μm wide, and 90 nm thick. Their spring constant was 0.1–0.2 N/m,and their resonant frequency and quality factor in an aqueous solution were 0.7–1 MHz
and ~2, respectively. For AFM imaging, the free oscillation amplitude was ~1 nm andthe set-point amplitude was around 90% of the free oscillation amplitude. The tapping
force estimated was less than 30 pN (27). An amorphous carbon tip was grown on theoriginal tip by electron beam deposition (28). The tip length was adjusted to ~1 μm. The
tip was sharpened by plasma etching under argon gas (tip apex, ~4 nm in radius).
AFM imaging and image processing
All AFM observations were performed at room temperature (24–26 °C). We usuallyused a scan area of ~45 × ~22 nm2 with (100–150) × (50–75) pixels. The AFM images
were captured at frame rates of 10–12.5 fps. After taking images, we tracked a target
molecule using two-dimensional (2D) correlation analysis to compensate for the slow
drift of the sample stage position in the x- and y-directions. A 3 × 3 pixel-average filter
was applied to each tracked image to reduce noise. For the AFM images obtained undernucleotide-free and AMPPNP conditions in which the conformational change does notoccur, frame averaging was applied before the pixel averaging (Fig. S2). The AFM
images were displayed first with black-to-white color scale and then the color scale wasnonlinearly assigned to the height level to highlight the top-surface structure (Fig. S4).
Correlation analysis
To determine the time evolution of the conformational state of each β subunit, we
calculated a 2D correlation coefficient for its image in the following way. As shown in
Fig. S7, for each β we arbitrarily chose a reference frame and a region of interest (ROI)
so that the ROI in the reference frame fully contained an open-state β subunit. The 2D
correlation coefficient defined below was calculated frame-by-frame for each ROI. For
images obtained in AMPPNP, we chose a closed- or open-state β subunit as thereference. The 2D correlation coefficient is defined as,

Here,

and

are heights at pixel pointreference ROI of the reference frame, respectively.
4

in a to-be-analyzed ROI and aandare mean values of theheight matrices

and, respectively.

Measurement of cumulated number of CCO shifts
As mentioned in the main text, the most populated conformational state was the CCO
state, in which one β subunit is in the open (O) state and the other two are in the closed(C) state. The other conformational states (COO, CCC, OOO) were much less frequentand therefore considered to be unusual conformations. To reveal unidirectional
propagation of the conformational states, we defined the cumulated number of the CCO
shifts (NCCO) as follows. When the CCO state first appears in successive AFM images,
NCCO is set to be 1. Then, when the CCO state shifts counterclockwise, NCCO isincreased by 1. In contrast, when the CCO state shifts clockwise, NCCO is reduced by 1.
Here, unusual states are ignored and therefore do not affect NCCO. This counting was
carried out for all consecutive frames.
Measurement of cumulated rotational angle
The open-state β had a higher protrusion than that of the closed-state β (Fig. 1). We
obtained the pixel position that has the highest intensity in each frame (P), and tracked
such positions in consecutive images (see inset of Fig. 2B). The averaged position of the
tracked highest-intensity positions was defined as the center (O). The cumulated
rotational angle of therelative to the vector
in the first frame was thencalculated.
Calculation of the degree of underestimation of unidirectionality
In the experiment shown in Fig. 2 and 3, the frame capture time (t) was 80 ms and thetime constant (τ) of the open state at 2 μM ATP was 0.66 s (Fig. S11) irrespective ofcounterclockwise or clockwise shift. Assuming a Poisson process, the probability (P(N))
that the shifts occur N-times within the frame capture time can be calculated byfollowing equation.
P(N) = (1/N!) × (t/τ)N × exp(–t/τ)Thus, the probability of single clockwise shift within the frame capture time is(1/1)×(0.08/0.66)×exp(–0.08/0.66) = 0.107, and that of two consecutive
counterclockwise shifts is (1/2)×(0.08/0.66)2×exp(–0.08/0.66) = 0.0065. Using thesevalues, fraction of the two consecutive counterclockwise shifts included in the apparent
single clockwise shift was estimated to be {0.0065/(0.0065+0.107)} ×100 = 5.7%.
5Simulation of AFM image
We used software (SPM simulator, Advanced Algorithm Systems Co., Tokyo, Japan) tosimulate AFM images of the α3β3 attached to a substrate surface at either the C- or
N-terminal side. The simulation was carried out with a simple hard-sphere model. The
cantilever tip was modeled as a circular cone (apex angle, 10°) with a small sphere(radius, 0.5–2 nm) at the apex. Crystal structures of the nucleotide-free α3β3 subcomplex(PDB code, 1SKY) and nucleotide-bound α3β3γ subcomplex (PDB code: 1BMF) wereused as the samples. For the latter case, the γ subunit was removed from the crystal
structure. Each atom in the protein was modeled as a hard sphere with a corresponding
van der Wars radius. We simulated AFM images using various radii for the tip-apex
sphere and found that a radius of 0.5 nm produced the images most similar to the actual
AFM images. The simulated images were processed by a low-pass filter with a cut-off
wavelength of 2 nm, because the spatial resolution of the AFM image wasapproximately 2 nm judging from the 2D Fourier transformation of actual AFM images.

LEGENDS FOR SUPPORTING FIGURES
Fig. S1. Purification and ATPase activity of α3β3 subcomplex. (A) SDS-PAGE of
purified α3β3 and α3β3γ subcomplexes. (B) ATP hydrolysis rate of α3β3 as a function of
ATP concentration. The rate obeyed Michaelis-Menten kinetics (solid line), yielding a
Michaelis constant (Km) of 12 μM and a maximum velocity (Vmax) of 12 s-1.
Fig. S2. Comparison of raw, temporally filtered, and spatially filtered AFM images. (A),(B), and (C) correspond to the raw, frame-averaged (22 frames), and 3 × 3
pixel-averaged images obtained without nucleotide, respectively. Scan range, 16 nm ×
16 nm. Pixel numbers, 46 pixels × 46 pixels. (D) and (E) correspond to the raw andpixel-averaged images in 2 μM ATP. Scan range, 14 nm × 14 nm. Pixel numbers, 32
pixels × 32 pixels.
Fig. S3. Wide-area AFM images of α3β3 subcomplex without nucleotide and in 2 mM
ATP. Frame rate, 1 fps. Scale bar, 20 nm. (A) An AFM image without nucleotide. In
this image, all molecules show a pseudo-six-fold symmetric ring shape in which threeβs in the ring are in the open state. (B) An AFM image in 2 mM ATP. Because the rateof conformational change was high in this ATP concentration as compared with the6frame rate of observation, all molecules showed a triangle shape in which βs in the ringare almost in the closed state (66% of a frame capture time) during scanning. These
results indicate that almost all of α3β3 molecules behave in the same way and show
different conformations depending on the presence and absence of ATP.
Fig. S4. Three dimensional illustrations of the crystal structure and AFM image innucleotide-free condition. The α and β subunits of the crystal structure are colored incyan and pink, respectively. The C-terminal DELSEED motif of β corresponding to thehigh protruding portions is highlighted in red. The color scale to display the AFM image
is not linearly set for the height level. This is because the height of α3β3 is about 9 nm
but the height variation at the top-most surface is less than 1 nm. To highlight the
top-most structure contained in the AFM image, the complete dark scale is used for theheight regions from the bottom to 80% of the highest position. Above this threshold, a
linear brightness scale is used.
Fig. S5. Experimental and simulated AFM images of the N-terminal side of α3β3
subcomplex (Online movie S2). (A) An AFM image experimentally obtained without
nucleotide. (B) N-terminal side of the atomic structure of α3β3 subcomplex (PDB code:
1SKY). The α and β subunits are colored in cyan and pink, respectively. (C) Simulated
AFM image constructed from the atomic structure (PDB code: 1SKY).
Fig. S6. Tight correlation between two types of conformational changes in β observed in
ATP; different heights of protrusion (“H,” high; “L,” low) and open (O)/closed (C)conformations of the distal region. The higher protrusion is mostly accompanied by theopen conformation, whereas the lower protrusion is mostly accompanied by the closed
conformation.
Fig. S7. Reference AFM images and regions of interest (ROIs) used in the correlation
coefficient analysis of β conformations observed in ATP (results shown in Fig. 2). The
transparent boxes show ROIs. The image of β in each ROI appears to be in the openstate.
Fig. S8. Correlation coefficient analysis of three βs in α3β3 subcomplex observed innucleotide-free condition. (A) AFM image of α3β3. (B) Reference images and ROIs
used in the correlation analysis. For each β, an arbitrary image was chosen as thereference image. (C) Correlation coefficient distributions for the three βs. (D) Time7

courses of correlation coefficient for three βs.
Fig. S9. Correlation coefficient analysis of three βs in α3β3 subcomplex observed in
AMPPNP. (A) AFM image of α3β3. (B) Reference images and ROIs used in thecorrelation analysis. Reference images were chosen so that β1 appears to be in the openstate whereas β2 and β3 appear to be in the closed state. (C) Correlation coefficient
distributions for the three βs. (D) Time courses of correlation coefficient for the three βs.
Fig. S10. Distributions of the number of successive shifts of the “CCO” state inclockwise (top) and counterclockwise (bottom) directions. Distributions at (A) 2 μM,(B) 3 μM, and (C) 4 μM ATP. The direction of the “CCO” shift is clearly biased in the
counterclockwise direction. The values for “mean” indicate the average number ofsuccessive shifts, and the values for “s.d.” indicate the standard deviations.
Fig. S11. Histograms of dwell times for the open (top) and closed (bottom) states invarious concentrations of ATP (A: 2 μM, n = 266; B: 3 μM, n = 426; C: 4 μM, n = 374).
Dwell time distributions for the open state are well fitted with single exponential decayfunctions (solid lines on top). The distributions for the closed state are fitted withfunctions representing consecutive reactions with two identical time constants:
A × t × exp(–t/τ) (solid lines on bottom).
Fig. S12. Loss of a single subunit stops rotary propagation of conformational changes.
(A and D) AFM images before and after loss of β1 (A) or α1 (D) (Online movies S7 andS8). (B and E) Time courses of correlation coefficients for the three βs designated in (A)and (D), respectively. Solid horizontal lines show the mean correlation coefficients forthe periods of open and closed states. The states are judged by examining whether thecorrelation coefficients are above or below a threshold (0.978). β1 (B) is lost at 16.2 s
and α1 (E) is lost at 9.4 s (vertical broken lines). (C and F) Cumulated angles of open βmeasured using the highest pixel position in each frame. [ATP], 2 μM; frame rate, 12.5
fps.

8

A

α3β3γ

Marker

α3β360 kDaαβ50 kDa40 kDaγ
30 kDaATP hydrolysis rate (s-1)

B

α3β3

10

Km = 12 μM
Vmax = 12 s-1
1

1

10

100

ATP concentration (µM)

Fig. S1

1000

A

B

D

E

C

Fig. S2

A

Nucleotide Free
20021_33

B

2 mM ATP8.9 nm

8.7 nm

20nm

20nm
0 nm

Fig. S3

0 nm

9.3 nm

9.3
17
nm

nm
0

0

0
nm

Fig. S436

0 nm

9.1 nm

A

5 nm
0 nm

B

C

αE
βE

βE

αE

αE

βE

Fig. S5

61.8 %

2000

32.5 %

1500
1000

&

Fig. S6

C

3

&

C

2

O
&

&

1

L

0

O

-1

0.6 %

L

5.1 %

H

500

H

Number of events

2500

4

β1

β3
β2

Fig. S7

A

B

β3
β1
β2

80

β3

0
100
50

Open
β2

0
100
50

D

Open

160

Correlation coefficients

Numbers of frames

C

Open
β1

1.0

0
0.92

0.94

0.96

0.98

β3

0.9

1.00

1.0

β2

0.9
1.0

β1

0.9
0

10

20

Time (s)Correlation coefficients

Fig. S8

30

40

A

B

β1

β2

β3

C
β3

0
10

Correlation coefficients

10

Numbers of frames

D

ClosedClosed
β2

0

Open

10

β1

β3

0.9
1.0

β2

0.9
1.0

β1

0.9

0
0.92

1.0

0.94

0.96

0.98

1.00

0

1

2

Time (s)Correlation coefficients

Fig. S9

3

4

5

B

[ATP] = 2 μM
40

Clockwise

Number of events

Number of eventsA

mean = 1.1

20

s.d. = 0.3
015Counterclockwise

10

mean = 4.7

5

s.d. = 5.0[ATP] = 3 μM
80
60

Clockwise40

mean = 1.2

20

s.d. = 0.6

0
40
30

Counterclockwise

20

mean = 3.1

10

s.d. = 2.4

0

0
0

5

10

15

20

25

30

0

[ATP] = 4 μM

Number of events

40

Clockwisemean = 1.2

20

s.d. = 0.5

0
16
12

Counterclockwise

8

mean = 4.7

4s.d. = 3.9

0
0

5

10

15

20

25

10

15

20

25

Number of successive sifts of “CCO”

Number of successive sifts of “CCO”

C

5

30

Number of successive sifts of “CCO”

Fig. S1030

B[ATP] = 2 μM

30
20

Open
mean = 0.66 s

10

τ = 0.67 s

015
Closed10

mean = 1.30 s

50

τ = 0.65 s

0

1

2

3

4

[ATP] = 3 μM

80

Number of events

Number of events

A

5

6

60
40

40

Number of events

40

mean = 0.35s

20τ = 0.26 s

040
Closedmean = 0.71 s
τ = 0.31 s

0

0

1

2

3

4

mean = 0.82 s
τ = 0.40 s

0

1

2

3

4

Dwell time (s)020Closed

20

80
Open

τ = 0.33 s

0[ATP] = 4 μM

60

mean = 0.41 s

20

Dwell time (s)

C

Open

5

6

Dwell time (s)Fig. S11

5

6

A

D

β1

α1
β2

5 nm

Correlation coefficients

0.8

β3

0.6
1.0
0.8

β2

0.6
1.0
0.8

β1
0

5

10

Cumulated angle (degrees)

15

Time (s)

20

25

5 nm

β3

0.6
1.0
0.8

β2

0.6
1.0
0.8

30

β1

2160

1080

5

10

15

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

F

3240

0

0.8

0.6

C

0

α2

E 1.0

20

25

30

Cumulated angle (degrees)

Correlation coefficients

B 1.0

0.6

α3
β3

β3

β2

β1

Time (s)

1440
1080
720
360
0

Time (s)Fig. S12Time (s)Table S1. Fraction of molecules showing conformational changes undervarious nucleotide conditions.
Number ofFraction ofchange-detectedchange-detected

molecules

molecules

53

0

0%

1 mM AMPPNP

46

0

0%

2 μM ATP

95

90

89%

3 μM ATP

86

81

94%

4 μM ATP

64

62

97%

Nucleotide

Number of

conditions

observed molecules

Nucleotide-freeReferences and Notes
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