Articles
https://doi.org/10.1038/s41565-020-00798-9

Structural and dynamics analysis of intrinsicallydisordered proteins by high-speed atomic force
microscopy
Noriyuki Kodera1,8, Daisuke Noshiro 1,8, Sujit K. Dora2,8, Tetsuya Mori2, Johnny Habchi3,David Blocquel3, Antoine Gruet3, Marion Dosnon3, Edoardo Salladini 3, Christophe Bignon3,
Yuko Fujioka4, Takashi Oda5, Nobuo N. Noda 4, Mamoru Sato5, Marina Lotti 6,
Mineyuki Mizuguchi7, Sonia Longhi 3 ✉ and Toshio Ando 1 ✉Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles indiverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering theirstructural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly foldedand constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the numberof amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

I

DPs constitute nearly a half of the entire protein realm1,2 and function as hubs in signalling and regulation of transcription, translation and cell cycle3. Our understanding of IDPs, however, falls far
behind that of well-structured proteins. X-ray crystallography andelectron microscopy have no practical application for IDPs because
intrinsically disordered regions (IDRs) hamper crystallization andare too thin to be visualized by electron microscopy. Nuclear magnetic resonance (NMR) spectrometry is so far most instrumental in
the structural analysis of IDPs4,5 but provides only distance restraints
between amino-acid (aa) residues separated by <2 nm. To complement NMR short-range structural information and estimate theoverall dimensions of IDPs, small-angle X-ray scattering (SAXS)
is used6. However, both are ensemble-averaging methods that cannot examine individual conformations and their populations. To
mitigate this problem, computational ensemble generation of disordered conformations is attempted by simulations7,8 or using statistical distribution of coil conformations from structure databases9,10.
Single-molecule Förster resonance energy transfer spectroscopy,
which can resolve structural and dynamic heterogeneity, can beused for the structural analysis of chemically denatured proteins and
IDPs11–13. Nevertheless, the donor and acceptor have to be in close
proximity (less than about 10 nm). Thus, we still lack versatile techniques enabling simultaneous and temporal assessment of both local(even with semiresidue resolution) and overall structures of IDPs.
Here we explored the potential of HS-AFM14,15 for structural anddynamics analysis of IDPs. All imaging experiments were performedin amplitude modulation mode, where a cantilever was verticallyoscillated at its resonant frequency of around 1 MHz in buffer solution.
The cantilever free oscillation amplitude and the set point amplitude

were set at appropriate values so that the loss of cantilever’s oscillationenergy per tap was adjusted to be 2–3 kBT on average (kB, Boltzmann
constant and T, room temperature in kelvin) (Supplementary Note 1).
This energy, which is considered to be transferred to the specimen, ispartitioned into many degrees of freedom in the specimen and dissipates quickly into solution. As demonstrated previously for an IDP,
the facilitates chromatin transcription (FACT) protein, HS-AFM canvisualize the thin and flexible structure of IDRs16,17. These studies,along with numerous previous HS-AFM studies performed on structured proteins, for example, refs. 18–21, have ruled out possible effectsof the tip-sample contact on the structural properties and functionof the protein under study. The present study on various IDPs showsthat HS-AFM can identify the fully disordered regions and the border between disordered and folded regions, at the near residue level.
Moreover, it provides information on the structural transition dynamics and the structural nature of transiently folded conformations.

Structural analysis of PQBP-1 constructs

We first imaged polyglutamine tract binding protein-1 (PQBP-1)
and its four deletion constructs with shorter IDRs placed on mica,
at 13.1–23.8 frames per second (fps) using HS-AFM (Fig. 1 andSupplementary Table 1). The HS-AFM video (Supplementary
Video 1) revealed that wild-type (WT) PQBP-1 has a small globule
at one end and a long flexible, thin structure (that is, IDR) (Fig. 1b,top), consistent with previous NMR data22. The N-terminal segment (1–47), for which no NMR data are available, appeared to bepart of the globule containing the ordered WW domain (48–81).
In the five constructs (Fig. 1a), the IDRs with average diameter
0.5 ± 0.1–0.3 nm (mean ± s.d.) (Fig. 1e and Supplementary Table 1)

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa, Japan. 2Department of Physics, Institute of Science andEngineering, Kanazawa University, Kakuma-machi, Kanazawa, Japan. 3Aix-Marseille University and CNRS, Laboratoire Architecture et Fonction desMacromolecules Biologiques (AFMB), UMR 7257, Marseille, France. 4Institute of Microbial Chemistry, Tokyo, Japan. 5Graduate School of Medical Life
Science, Yokohama City University, Yokohama, Japan. 6Department of Biotechnology and Biosciences, State University of Milano-Bicocca, Milano, Italy.
7
Faculty of Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 8These authors contributed equally: Noriyuki Kodera, Daisuke Noshiro,
Sujit K. Dora. ✉e-mail: sonia.longhi@univ-amu.fr; tando@staff.kanazawa-u.ac.jp
1

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

181

Articles

NATurE NAnoTEcHnology

82 104164 190

WWD
PRD
184 aaWT PQBP-1 (1–265)
133 aaPQBP-1 (1–214)

1

PQBP-1 (1–164)1

214

3.74 s

4.53 s

130

0

D

P2

19.54 sPQBP-1 (1–164)

H2
Mica

H2/2

e

WT PQBP-1 (1–265)
WWD
N = 1,360, Mol = 4〈H1〉 = 1.9 nm
s.e.m. = 0.002 nm

22.31 s022.44 s1.37 s18.48 s23.06 s1

H1 (nm)2

3

18.09 s18.30 s22.11 sNumber of frames

0

0

10

0

1

H2 (nm)2

6.26 s6.80 sNumber of frames

1.83 s

30

40

0

10

20
R2D (nm)

3040

PQBP-1 (1–134)
N = 1,178, Mol = 3〈R2D〉 = 9.3 nm
s.e.m. = 0.07 nm

200

0

10

20
R2D (nm)

3040

PQBP-1 (1–114)

N = 925, Mol = 4〈R2D〉 = 7.1 nm
s.e.m. = 0.02 nm

200

0

3

20

R2D (nm)

PQBP-1 (1–164)
N = 1,190, Mol = 3〈R2D〉 = 11.0 nm
s.e.m. = 0.04 nm

400
WT PQBP-1 (1–265)
Tail-end
N = 1,360, Mol = 4〈H2〉 = 0.5 nm
s.e.m. = 0.005 nm

PQBP-1 (1–114)

Number of frames

0

40

160

0

700

350

30

PQBP-1 (1–214)
N = 1,753, Mol = 4〈R2D〉 = 14.4 nm
s.e.m. = 0.14 nm

400

PQBP-1 (1–134)

Number of frames

0

20

180

0

250

10

320

H1

500

0

360

R2D

d

WT PQBP-1 (1–265)
N = 1,360, Mol = 4〈R2D〉 = 17.2 nm
s.e.m. = 0.09 nm

134

c

H1/2

260

R2D (nm)

114
33 aa
PQBP-1 (1–114)

P1

1.07 sPQBP-1 (1–214)

1

PQBP-1 (1–134)

CTD

164

83 aa

53 aa

265

Number of frames

1

48

f

Number of frames

1

WT PQBP-1 (1–265)b

Number of frames

a

0

10

20
R2D (nm)30

40Fig. 1 | Structural features of PQBP-1 constructs revealed by HS-AFM imaging. a, Domain diagrams of five PQBP-1 constructs containing IDRs (light blue),folded WWD (grey) and the N-terminal segment not investigated with NMR (yellow). b, HS-AFM images and schematized molecular features of PQBP-1
constructs. Z scale, 0–3.5 nm. Top two scale bars, 30 nm; lower three scale bars, 20 nm. c, Schematics showing the observed molecular characteristics of
the PQBP-1 constructs (top, top view; bottom, side view): grey spheres, N-terminal globular domain; blue thick solid lines, IDRs; dashed red lines, schematic
topographies of the globular domain convoluted with the finite size of the tip apex in lateral contact with the globule. d,e, Height distributions of globule (d)and tail end (e) in WT PQBP-1. f, R2D distributions of tail-like segments in PQBP-1 constructs. Solid blue lines show most probable fitting curves. The values
obtained by Gaussian distribution fitting are mean ± standard error of the mean (s.e.m.). This definition is the same for all Gaussian fitting results in thispaper, unless otherwise stated. Mol in d–f indicates the number of molecules analysed. This is the same for Figs. 3 and 4.

appeared constantly disordered. To measure the two-dimensional(2D) end-to-end distance (R2D) of the IDRs, the direct distance
D between the N-terminal globule and the C-terminal end wasmeasured, followed by measurements of the N-terminal globuleheight H1 and the C-terminal end height H2. R2D was estimated as
R2D = D − H1/2 − H2/2 (Fig. 1c and Supplementary Note 2).
182

The estimated values of 〈R2D〉, 〈H1〉 and 〈H2〉 of PQBP-1 (1–214)
were independent of the imaging rate within the tested range(6.7–50 fps; Extended Data Fig. 1 and Supplementary Table 2), indicating no notable structural deformation of the molecule by its contact with the tip. Moreover, in WT PQBP-1 the C terminus position
was circularly distributed around the WW domain (Fig. 2a), andNature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology
a

d

40

e

3

f

6

2

0
–20

2

R2D, PQBP-1 (1–265)
Frame rate: 42 ms per frame
λ = 37 ± 7 s–1

1
0

R2D, PQBP-1 (1–214)
Frame rate: 33 ms per frame
λ = 62 ± 10 s–1

4
3
2
1

Autocorrelation

Autocorrelationy (nm)

20

Autocorrelation

5
R2D, PQBP-1 (1–214)
Frame rate: 20 ms per frame
λ = 112 ± 26 s–1

1

0

0
40

g
200

Δt
+13.5 nm–13.5 nm

0
200

2Δt
+13.8 nm

0200
–13.9 nm3Δt+13.6 nm–13.6 nm4Δt+14.3 nm–13.9 nm5Δt+14.0 nm

0

0
–80

–40

0

40

80

x Displacement (nm)j

H2, NTAIL-GFP
λ = 7.3 ± 0.8 s–1

3
2
1

3

–1

4

i

6

5
H2, NTAIL-Trx, pH 6.0
λ = 7.5 ± 0.6 s–1

4
3
2
1

3
2
1
0
–1

6

10

0

2

4
6
Time (s)8

H2, NTAIL-Trx, pH 7.0
λ = 7.2 ± 0.6 s–1

4

–1
8

3

6

–1
4
6
Time (s)2Time (s)

0
2

1

0

0

10

2

4
6
Time (s)810

H2, Trx-NTAIL, pH 6.0
λ = 3.1 ± 0.2 s–1

4
3
2
1
0

k

0

2

4
6
Time (s)

8

10

l

6

m 6

6
5

5
R2D, PNT-GFP
λ = 2.5 ± 0.1 s–1

4
3

Autocorrelation

20

1
2
3
4
5
Frame number difference (n)2Time (s)

0

30

10

1

0

5

4

–1

0

–1

5

5

Autocorrelation

Averaged absolute valuesof displacement (nm)

c

4

h

0

200

2
3
Time (s)60200Autocorrelation

Number of frames–13.6 nm15Autocorrelation

b

0

2
1

5
Autocorrelation

020
x (nm)

Autocorrelation–1–20

Autocorrelation

–40
–40

H2, Sic1-GFP
λ = 1.7 ± 0.1 s–1

4
3
2
1

3
2
1

0

0

0

–1

–1

–1

0

2

4
6
Time (s)

8

10

0

2

4
6
Time (s)8

10

R2D, Sic1-GFP
λ = 1.9 ± 0.1 s–1

4

0

2

4
6
Time (s)810Fig. 2 | Absence of directional bias by scanning tip and of notable impediment by tip and mica to diffusional motion of IDR, and autocorrelation functions
for time-series R2D(t) or H2(t) data. a, Two-dimensional scatter plots of the C-terminal end position relative to the WWD (the cross mark) in WT PQBP-1.
The red circle indicates the mean direct distance between the WWD and the C-terminal end (that is, radius of 〈R2D〉 + 〈H1〉/2 + 〈H2〉/2). b, Distributionsof C-terminal end displacements in the x direction occurred during time periods of nΔt (n = 1, 2, 3, 4, 5), where Δt is one frame acquisition time (42 ms).
The numbers shown on each panel are the mean displacements in the +x and −x directions. c, Averaged absolute values of displacements in the x (lightblue) and y (pink) directions, during time periods of nΔt. The thin vertical lines represent s.d. The large s.d. values reflect the flexible nature of IDR. d–m,
Autocorrelation functions of time-series data of R2D(t) (d–f, k, m) for IDRs contained in PQBP-1 (1–265) (d), PQBP-1 (1–214) (e,f), PNT–GFP (k) and Sic1-GFP(m), and autocorrelation functions of H2(t) data (g–j,l) for Box1 in NTAIL-GFP (g), NTAIL-Trx at pH 6.0 (h) and NTAIL-Trx at pH 7.0 (i), Box2 in Trx-NTAIL at pH 6.0(j) and the N-terminal collapsed structure in Sic1-GFP (l). In the case of the constantly disordered IDRs in the PQBP-1 constructs, the characteristic time for
the C-terminal end movement relative to the N terminus is much shorter than the one frame acquisition time (20 ms).

its displacement in the fast scan (x) direction occurring during different time intervals were symmetrically distributed around theirzero mean displacement (Fig. 2b). The averaged absolute values ofdisplacements in the x direction were nearly identical to those in
the slow scan (y) direction (Fig. 2c). Since the HS-AFM images were
captured during tip scanning relative to the sample stage from the−x to +x direction, these data showing no directional bias in the C
terminus displacements ascertain no effect of tip-molecule contact
on the IDR motion. Besides, the autocorrelation functions of timeseries of R2D showed no correlation of its change over time evenwhen the HS-AFM images were captured at 50 fps, suggesting no
impediment of the mica surface and tip to IDR diffusional motion
on this time scale (Fig. 2d–f).

The 〈R2D〉 values for these constructs were 17.2 ± 0.09,
14.4 ± 0.14, 11.0 ± 0.04, 9.3 ± 0.07 and 7.1 ± 0.02 nm (mean ± s.e.m.,
Supplementary Note 3) in the order of decreasing construct length(Fig. 1f), approximately following a scaling law 〈R2D〉 = β2D × Naaνwith β2D = 1.18 ± 0.11 nm (best fit value ± s.e.m.) and ν = 0.51 ± 0.02.
This Flory exponent ν is close to 0.5, which holds for 2D andthree-dimensional (3D) ideal and Gaussian chains23, indicating thatthese constantly disordered segments have similar values of persistence length (Lp) that can be estimated from




2Lp
L
;
hR22D ðLÞi ¼ 4Lp L 1 �
1 � exp �
2Lp
L

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

ð1Þ
183

Articles

NATurE NAnoTEcHnologya1

MIT1/MIT2

e

898
913

P1

P2

D

FLAG H6
R2D

3.5 s

10.0 s6.7 s15.9 s17.7 s359–389424–436

d

Mica

10

9.2 s

500

H1 (nm)02

H2 (nm)

300

0

10

20

R2D (nm)

0

6

k

4

30

40

0

1

1,600

6

Atg1
50 mM NaCl, pH 8.0
N = 1,926, Mol = 3
1st: 〈R2D〉 = 13.0 nm
s.e.m. = 1.04 nm
2nd: 〈R2D〉 = 20.5 nm
s.e.m. = 1.42 nm
F-test: P < 0.05

150

0

4

Atg1
MIT1/MIT2
50 mM NaCl
pH 8.0N = 1,926
Mol = 3
〈H2〉 = 3.2 nm
s.e.m. = 0.005 nm

250

0

2

Atg13
HORMA
100 mM NaCl
pH 7.0N = 3,674
Mol = 3
〈H1〉 = 2.5 nm
s.e.m. = 0.005 nm

600

l

0

n

0

1

2
H1 (nm)2

H2 (nm)

3

200

0

15

8

8
8
50 mM 300 mM
NaCl
NaCl

Atg13
Weighted mean = 28.0 nm
Weighted s.e.m. = 2.0 nm

20

6

5

7

8

pH

9

9

30

4

30

R2D (nm)

Atg1

20

3

45

6

FACT
(10SA)
10

NTAIL

4

Atg13
100 mM NaCl, pH 7.0
N = 3,345, Mol = 3
1st: 〈R2D〉 = 20.7 nm
s.e.m. = 0.51 nm
2nd: 〈R2D〉 = 28.1 nm
s.e.m. = 4.62 nm
F-test: P < 10–3

400

0

7
20 mM KCl

Atg13

Atg13
Tail-end
100 mM NaCl
pH 7.0N = 3,345
Mol = 3
〈H2〉 = 0.5 nm
s.e.m. = 0.004 nm

800

6Number of points

0

1,200

〈R2D〉 (nm)Atg1
Kinase domain
50 mM NaCl
pH 8.0N = 1,926
Mol = 3
〈H1〉 = 3.3 nm
s.e.m. = 0.008 nm

225

0

Number of frames

450

pH
Salt

30

10

j

Atg1
Weighted mean = 20.5 nm
Weighted s.e.m. = 0.9 nm

m 40

30.0 s

16.8 sNumber of frames

Number of frames
Number of frames

h

20

H2/2

〈Rg〉 (nm)Number of frames

g

6.8 s

641–661 H6

30〈R2D〉 (nm)

2.9 s

f

477 aai〈R2D〉 (nm)

HORMA
Atg13

739
744

460–521

268

H2

H1
H1/2

1

Atg13 (100 mM NaCl, pH 7)

255 aa587

Number of frames

Atg1 (50 mM NaCl, pH 8)cKinase

Atg1

b

332

0

0

100

200

3

300

N = 647
Mean = 0.5 nm
s.e.m. = 0.005 nm

150

0

0

Naa

0.5
1.0
Height (nm)

300

400

1.5

0
500

60

Fig. 3 | Structural features of Atg1 and Atg13 revealed by HS-AFM imaging and 〈R2D〉–Naa and 〈Rg〉–Naa relationships. a,c, Domain diagrams of Atg1
(D211A) (a) and Atg13 (c); the top and bottom red bars in c are Atg17- and Atg1-binding regions, respectively. b,d, HS-AFM images and their schematics
of Atg1 (D211A) (b) and Atg13 (d). Z scale, 0.0–3.5 nm. Scale bars, 30 nm. Light-blue arrow heads (d, 9.2 s and 30.0 s) indicate temporarily appearing
small globules. e, Schematics of observed molecular features of Atg1 (D211A) (top, top view; bottom, side view). f,g,j,k, Height distributions of kinase (f),MIT1/MIT2 (g) and HORMA (j) domains and C-terminal end of Atg13 (k). h,l, R2D distributions of Atg1 (D211A) (h) and Atg13 (l). i, Dependence of 〈R2D〉
in the longer state of Atg1 (D211A) on pH and salt type and concentration. m, pH dependence of 〈R2D〉 in the longer state of Atg13. n, 〈R2D〉–Naa relationship(blue line) from 〈R2D〉 data of PQBP-1 (closed blue circles), FACT (IDR in the SSRP1 subunit)17, NTAIL, Atg1 (D211A) and Atg13, and 〈Rg〉–Naa relationship fromSAXS data of IDRs in PQBP-1 (closed red circles). The 〈R2D〉 values for the Atg proteins are those in the longer states. Inset: height distribution of constantly
disordered IDR in WT PQBP-1.

184

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology
where L represents the contour length of a chain24. For an unstructured polypeptide chain, L = (Naa – 1) × 〈daa〉, where Naa is the numberof aa contained in the chain and 〈daa〉 is the average distance between
two adjacent aa, with the most likely value of 〈daa〉 being 0.36 nm(ref. 25). The Lp values estimated for these constructs were 1.16, 1.15,
1.11, 1.35 and 1.45 nm in the order of decreasing construct length.
The chain conformations of these IDRs are very likely to beaffected to some extent by their confinement to the liquid–micainterface because of their prominently flexible nature. To assess thisinfluence, we performed SAXS measurements in solution (that is,
not on mica) using four IDRs of PQBP-1 (82–265, 82–214, 82–164
and 82–134), yielding values of radius of gyration 〈Rg〉 of 3.62 ± 0.05,
3.10 ± 0.18, 2.69 ± 0.04 and 2.14 ± 0.04 nm, respectively (red circles
in Fig. 3n, Extended Data Fig. 2 and Supplementary Table 3). In
Gaussian chains, 〈R2D2〉 is theoretically identical to 2 × 〈R3D2〉,
where R3D is the end-to-end distance in solution, and the value of〈R3D2〉/〈Rg2〉 is 6.0 when L » Lp (ref. 26). Therefore, we expect 〈R2D2〉/〈Rg2〉 ≈ 12.0. However, the 〈R2D2〉/〈Rg2〉 values of the fully disordered
IDRs were considerably larger than 12.0 (20.0 on average), indicating that the mica
surface expands the IDR 2D dimensions roughlypﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
by a factor 20=12 ≈ 1.3. Its possible cause could be frictional
forces locally Iexerted from mica against fast Brownian motion of
the IDR chain, which would increase the IDR chain’s undulation
wavelength and/or decrease the undulation amplitude, resulting inswelling of its 2D dimensions27.

Structural analysis of autophagy proteins

Next, we imaged autophagy proteins containing longer IDRs, Atg1
(D211A kinase-dead mutant) and Atg13 (Fig. 3 and Supplementary
Videos 2 and 3). The IDR of Atg1 (D211A) showed a double-Gaussian
distribution of R2D (Fig. 3h), suggesting interactions between the
IDR and the dead kinase domain, in agreement with phosphorylation of the IDR by the N-terminal kinase domain of the WT protein(Extended Data Fig. 3b). The IDR of Atg13 exhibited temporarily
appearing and disappearing small globules (Fig. 3d, 9.2 and 30.0 s),
resulting in a double-Gaussian distribution of R2D (Fig. 3l). Since thefour small regions within the IDR (red bars in Fig. 3c) are known tointeract with Atg1 or Atg17 (ref. 28), the small globules probably correspond to these regions. The 〈R2D〉 values in the longer IDR statesof Atg1 and Atg13 were 20.5 ± 1.42 and 28.1 ± 4.62 nm (Fig. 3h,l),respectively, and remained nearly unchanged upon pH increase
from 6 to 8 for both proteins and upon modification of both salt
type and concentration for Atg1 (Fig. 3i,m). In contrast, the 〈R2D〉
values in the shorter states varied (Extended Data Figs. 3 and 4 andSupplementary Tables 4 and 5). Since the IDRs in the longer states
are considered to be fully disordered, we estimated their Lp values
using (Naa, 〈R2D〉) = (255, 20.5 nm) for Atg1 and (Naa, 〈R2D〉) = (477,28.1 nm) for Atg13, yielding Lp of 1.18 and 1.17 nm, respectively.
These ‘measured Lp values’ close to each other are also close to thoseof the IDRs in the PQBP-1 constructs. The height of the C-terminalend of Atg13 was 0.5 ± 0.2 nm (mean ± s.d.) (Fig. 3k).

Structural analysis of NTAIL

Next, we observed the C-terminal domain of the measles virus(MeV) nucleoprotein (NTAIL). Its Box1 (401–420) and Box2 (486–502) regions were previously shown to correspond to a molecular

recognition element29,30 of helical nature (α-MoRE) in equilibrium
among completely unfolded and folded helical conformations30–34.
We used NTAIL constructs fused at their N or C terminus to green
fluorescent protein (GFP) or thioredoxin (Trx) to impede the otherwise very fast diffusion of NTAIL. NTAIL-GFP (Fig. 4a) showed athin and flexible tail-like structure and a C-terminal GFP globulewith mean height of 3.2 nm (ref. 35) (Fig. 4d,g and Supplementary
Video 4). The IDR showed a temporarily appearing and disappearing small globule at the N terminus corresponding to Box1
(Fig. 4d). Except for this, the IDR was observed to be fully disordered(with mean height of 0.4−0.5 nm) (Extended Data Fig. 5a). Box2
was indiscernible due to its close proximity to GFP but appeared in
the images of Trx-NTAIL (Extended Data Fig. 6, and Supplementary
Fig. 1 and Supplementary Video 5).
The R2D histogram of the IDR in NTAIL-GFP showed a single
Gaussian peak at 11.6 ± 0.1 nm (Fig. 4h and Supplementary Fig. 2),
consistent with the very small population of Box1 in the higher state,
as observed in both NTAIL-GFP (Fig. 4k) and NTAIL-Trx (Extended
Data Fig. 7f,i and Supplementary Video 6), and the large population of Box2 in the higher state as observed in Trx-NTAIL (Extended
Data Fig. 6e). From 〈R2D〉 = 11.6 nm and Naa = 88 in the IDRs spanning 421–485 and 503–525, Lp was estimated to be 1.16 ± 0.02 nm, avalue close to those described above. HS-AFM images of NTAIL-Trx
also gave similar 〈R2D〉 values at pH 6.0 and 7.0 (11.2 ± 0.1 and11.1 ± 0.1 nm, respectively) (Extended Data Fig. 7e,h), yielding Lpvalues around 1.06 nm. However, HS-AFM images of Trx-NTAIL
gave a slightly smaller value, 〈R2D〉 = 10.1 ± 0.1 nm (Extended Data
Fig. 6d), resulting in a smaller LP value (0.86 ± 0.02 nm), possibly due
to an increased order propensity of Box1 induced by interaction withthe adjacent Trx. To gain more insights into the structural transitionsof Box1 and Box2, we measured their height H2. The H2 histogramsof Box1 in NTAIL-GFP and NTAIL-Trx (at pH 6.0 and 7.0), as well asthat of Box2 in Trx-NTAIL, showed double-Gaussian peaks at 0.8 and1.1 nm (Fig. 4k, Extended Data Figs. 6e and 7f,i and Supplementary
Tables 6 and 7). Moreover, despite the single Gaussian distribution of
R2D, these H2 distributions were correlated with the corresponding R2D
distributions (Fig. 4n and Supplementary Fig. 3), suggesting that the
IDR length partly changes due to order–disorder transitions in these
globules. The order propensity of Box1 and Box2 was estimated fromthe population ratios (Ke) between their higher and lower states, yielding
Ke = 0.19–0.28 (depending on the pH value) and Ke = 1.91 (at pH 6.0),
respectively (Supplementary Tables 6 and 7). The H2 value of 0.8 nm islarger than the height of fully disordered regions, indicating that Box1
and Box2 are both partially ordered, even in their lower height states.
The H2 value of 1.1 nm is identical to the diameter of a single α-helix,
suggesting that Box1 and Box2 are α-MoREs, consistent with previous
studies29,32. From these features, the dynamic structure of NTAIL could
be sketched (Fig. 4q).

Power law for 〈R2D〉

In the above observations, we used proteins with IDR boundaries
previously identified at the residue level. The 〈R2D〉 values of these
IDRs in their fully disordered state were plotted against Naa (Fig. 3n),
together with a data point (Naa, 〈R2D〉) = (121, 14.3 ± 0.18 nm)
obtained by our previous HS-AFM study for a fully disordered IDR
in unphosphorylated FACT protein17. The nine points could be fitted

Fig. 4 | Structural and dynamic features of NTAIL, PNT and Sic1 revealed by HS-AFM imaging. a–c, Domain diagrams of NTAIL-GFP (a), PNT-GFP (b) andSic1-GFP (c). The numbers (top and bottom) are original aa sequential numbers and those of the constructs, respectively. d–f, HS-AFM images and theirschematics of NTAIL-GFP (d), PNT-GFP (e) and Sic1-GFP (f). Z scale, 0.0–3.5 nm. Scale bars, 30 nm. The light-blue arrow-head (e, middle) indicates a rarely
appearing tiny globule, likely to be α4. g, Height distribution of GFP in NTAIL-GFP. h–j, R2D distributions of NTAIL-GFP (h), PNT–GFP (i) and Sic1-GFP (j).
k–m, H2 distributions of N-terminal globules in NTAIL-GFP (k), PNT-GFP (l) and Sic1-GFP (m). n–p, Relationship between H2 and R2D for NTAIL-GFP(n), PNT-GFP (o) and Sic1-GFP (p). q–s, Schematics showing structural and dynamic features of NTAIL (q), PNT (r) and Sic1 (s). The top and bottom
panels correspond to the more- and less-ordered states, respectively. The numbers in red and blue represent the numbers of aa contained in the respective
small globules and fully disordered IDRs, respectively. The red and blue arrows indicate the kinetic nature of changes in H2 and R2D, respectively.
Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

185

Articles

NATurE NAnoTEcHnology
Structural analysis of the MeV phosphoprotein (PNT)

to a power law 〈R2D〉 = β2D × Naaν with β2D = 1.16 ± 0.057 nm andν = 0.52 ± 0.009 (blue solid line in Fig. 3n). The mean observed Lp
value was 1.18 nm (Supplementary Table 8).

1h

NTAIL-GFP

545

k

1,000

Number of framesd

NTAIL-GFP
N = 4,754, Mol = 11
〈R2D〉 = 11.6 nm
s.e.m. = 0.11 nm

500

0

10

0

20

30

NTAIL-GFP
1st: 〈H2〉 = 0.8 nm
s.e.m. = 0.03 nm
2nd: 〈H2〉 = 1.1 nm
s.e.m. = 0.82 nm
F-test: P < 10–4

600

0

40

0

1

Number of frames

Sic1-GFP

r

NTAIL
21
401
(8)

503
525
(110)
(132)
18

63
422
(29)
–1

kDO = 1.2 - 1.6 s

486
(93)

Ke = 1.91

kOD = 5.7 - 6.1 s–1

kOD = 1.1 s–1

401
(8)

422
(29)
21

486
(93)
63

18

1
(8)

20
30
R2D (nm)

α4

~53

525
(132)

H2 (nm)0

1
2
H2 (nm)Sic1~190(~197)

229
(236)

kDO

kDO = 2.0 s–1~1001
α1/2 (8)kOD = 0.7 s–1

kOD = 0.5 s–1
-

-

α4

= 1.1 s–1
Ke = 1.63

Ke = 3.73

-

0

10

229
(236)

~30
1
(8)

20

30

40

2
1

~95

10

0

20
30
R2D (nm)40

Sic1-GFP
Slope: –0.019 ± 0.001
Intercept: 1.43 ± 0.02 nm

3
2
1
0

3

PNT–GFP
Slope: –0.012 ± 0.001
Intercept: 1.18 ± 0.01 nm

0

10

20
30
R2D (nm)

40

284
(291)

kDO = 1.1 s–1

~190

A-IDR

1

0

p

225

5

NTAIL-GFP
Slope: –0.018 ± 0.001
Intercept: 1.08 ± 0.01 nm

3

3

Sic1-GFP
1st: 〈H2〉 = 0.9 nm
s.e.m. = 0.05 nm
2nd: 〈H2〉 = 1.3 nm
s.e.m. = 0.28 nm
F-test: P < 0.02

1
(8)

-

1
2
H2 (nm)

450

0

40

0

4

R2D (nm)PNT–GFP〈H2〉 = 1.1 nm
s.e.m. = 0.003 nms- A-IDR
~175

α3

186

10

PNT
α1/2

503
(110)

23

m

0

3

2

0

3

700

0

40

Sic1-GFP
N = 2,801, Mol = 5
1st: 〈R2D〉 = 12.2 nm
s.e.m. = 0.66 nm
2nd: 〈R2D〉 = 20.5 nm
s.e.m. = 2.14 nm
F-test: P < 0.02

225-

Ke = 0.19 - 0.28

2030
R2D (nm)

α323kDO = 2.1 s–1

10

450

0

q

0

2

2

H1 (nm)

3

o

1,400

Number of frames

Number of frames

PNT–GFPj

PNT–GFPN = 5,831, Mol = 4
1st: 〈R2D〉 = 8.9 nm
s.e.m. = 0.33 nm
2nd: 〈R2D〉 = 14.3 nm
s.e.m. = 9.26 nm
F-test: P < 10–20

700

0

f

l

1,400

1

H2 (nm)Number of frames

i

0

n

1,200

R2D (nm)

e

500

0

292
306

Sic1 (284 aa)

8490

NTAIL-GFP
N = 4,754
Mol = 11
〈H1〉 = 3.2 nm
s.e.m. = 0.004 nm

H2 (nm)

Sic1-GFP

1,000

386

H2 (nm)cg

Box2 526 TEV Box1: 401–420, Box2: 486–502
GFP (240 aa)
NTAIL-GFP
133
1
NTAIL (125 aa)
8147
α1/2: 1–37, α3: 87–93,
H6 α1/2
α3
α4A-IDR: 124–168, α4: 189–198
230
1
A-IDR
PNT–GFP
237
1
PNT (229 aa)
8
251
H6
1
285
401 Box1

Number of framesb

H6

Number of framesaAlthough the N-terminal domain of PNT is mainly disordered,
the regions 1–37, 87–93 and 189–198 were shown by NMR to have

~30
(~37)

Ke = 1.50
kOD = 0.8 s–1
~257
284
(291)

~128

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology
α-helical propensities, and hence are named α1/2, α3 and α4, respectively36. α3 and α4 are interspaced by an acidic IDR (124–168). The
resistance of the N-terminal 27–99 region of PNT to proteolysis
in the presence of trifluoroethanol37 (a secondary structure stabilizer33,34), cannot be rationalized from the NMR studies, thus suggesting the existence of a large-scale structure undetectable by NMR. To
shed light onto this issue, we imaged a PNT segment (1–229) fused
to GFP (PNT-GFP, Fig. 4b). PNT-GFP exhibited a thin and flexible
tail-like structure with a temporarily appearing and disappearing
small globule at the N-terminal end, together with the C-terminal
GFP (Fig. 4e and Supplementary Video 7). In addition, a small globule close to GFP could be occasionally detected (middle panel inFig. 4e), very probably corresponding to α4. The R2D histogram of
the IDR was best fitted to a double Gaussian with peaks at 8.9 ± 0.33
and 14.3 ± 9.26 nm (Fig. 4i). The mean height of the IDR was
0.4–0.5 nm, in both metastable shorter and longer states (Extended
Data Fig. 5b), indicating that the IDR is fully disordered even on
disorder-to-order transition of the small globule. Unlike R2D, the H2
histogram of the N-terminal small globule undergoing transitions
was best fitted to a single Gaussian with a peak at 1.1 ± 0.003 nm(Fig. 4l). To estimate Naa in the IDR, we used both the power law〈R2D〉 = 1.16 × Naa0.52 and 〈Lp〉 = 1.18 nm as obtained above, yielding
Naa = 52 and 129 from the power law, and Naa = 54 and 128 from〈Lp〉 for the shorter and longer states, respectively. Therefore, thenumber of aa contained in the small globule is roughly estimatedto be 175 and 100 for the shorter and longer tail states, respectively(α4 does not much affect the estimate). The number around 100
suggests that α1/2, α3 (87–93) and the IDR segment between them
(38–86) are contained in the small globule in both longer andshorter IDR states. Therefore, the region encompassing the N terminus and helix α3 always forms a compact structure, explaining theresistance to proteolysis of the N-terminal segment extending to aa
99. The number around 175 suggests that the structural transitions
between the two states mainly occur by association and dissociation between the N-terminal compact structure and the acidic IDR(124–168) (Fig. 4r). Note that 〈H2〉 = 1.1 nm possibly corresponds tothe height of α-helices contained in the small globule.

Structural analysis of Sic1

Finally, using Sic1-GFP (Fig. 4c) we studied this IDP that possesses
a propensity to adopt a collapsed chain-like form38. Sic1-GFP exhibited a thin and flexible tail-like structure with a temporarily appearing and disappearing small globule at the N-terminal end, together
with the C-terminal GFP (Fig. 4f and Supplementary Video 8).
The R2D histogram for the IDR was best fitted to a double Gaussianwith peaks at 12.2 ± 0.66 and 20.5 ± 2.14 nm (Fig. 4j). The IDR isfully disordered in both shorter and longer states, as judged fromits mean height of 0.4–0.5 nm in both metastable states (Extended
Data Fig. 5c,d). The H2 histogram for the N-terminal small globule
undergoing transitions was best fitted to a double Gaussian withpeaks at 0.9 ± 0.05 and 1.3 ± 0.28 nm (Fig. 4m). The height difference (0.4 nm) between the two states is small, compared to the large
difference (8.3 nm) between the two 〈R2D〉 values, suggesting thatthe partially folded state has a flattened, collapsed structure, consistent with a previous study38. As done above for PNT-GFP, we estimated Naa contained in the shorter and longer IDRs; we obtained
Naa = 95 and Naa = 259 from the power law and Naa = 95 and Naa = 255
from 〈LP〉 = 1.18 nm for the shorter and longer states, respectively.
Therefore, from the total number of aa contained in Sic1 (284), thenumber of aa contained in the small globule was estimated to beabout 30 and 190 for its lower and higher states, respectively. The
large number around 190 indicates that as much as about 65% of thewhole protein is loosely folded, with only the remaining C-terminal
region (encompassing about 95 aa) being fully extended. The
remarkable difference between 30 and 190 indicates that a large segment containing about 160 residues undergoes a transition between

fully unstructured and loosely folded conformations (Fig. 4s). The
folding propensity derived from the H2 distribution (Ke = 1.6) isclose to that estimated from the R2D distribution (Ke = 1.5).

Order propensity and structural transition dynamics

From all Ke values obtained above, we can infer, for all small
globules undergoing transitions, the following relative orderpropensity: PNT > Box2 (NTAIL) > Sic1 » Box1 (NTAIL) (Fig. 4q–s
and Supplementary Tables 6 and 7). Disorder predictions failed
to detect these subtle differences (Supplementary Figs. 4 and 5),
underlining the importance of experimental analysis of order–
disorder transitions. The order propensity and the transiently
ordered structures very probably dictate key features in bindingof these IDPs to their targets (Supplementary Note 4). Next, we
calculated autocorrelation functions G(τ) for time-series H2(t) or
R2D(t) data of these IDPs to estimate the order-to-disorder anddisorder-to-order transition rates (kOD and kDO, respectively).
The rate constants can be expressed as kOD = λ/(1 + Ke) andkDO = λKe/(1 + Ke), where λ ≡ kOD + kDO is the decay rate of G(τ). All
G(τ) were best fitted to single exponential functions (Fig. 2g–m).
Time correlations in the G(τ) for R2D(t) (Fig. 2k,m) reflect theorder–disorder transitions, because Brownian undulation motionof constantly disordered IDRs in PQBP-1 constructs is too fast
to appear in G(τ) (Fig. 2d–f). In fact, in Sic1 the G(τ) for H2(t)and R2D(t) were nearly identical (Fig. 2l,m). In contrast to thewidely varying lifetime of the ordered state (0.16–1.89 s) among
these IDPs, the lifetime of disordered state does not vary much
(0.49–0.94 s) (Fig. 4q–s and Supplementary Table 6).

Refinement of mica effect, power law and Lp

SAXS data of non-canonical IDPs obtained by truncation of foldable
proteins yielded ν = 0.54, despite their hydrophobic nature39. SAXS
data of chemically denatured proteins yielded ν = 0.598 (ref. 40),
indicating that chemically denatured proteins are more extended
than native IDRs11,41 and behave as polymer chains in good solvent(theoretically ν = 0.6, ref. 23, or 0.588, ref. 42). In contrast, 〈Rg〉 versus Naa plots for 72 naturally occurring IDPs without permanent
secondary structures were found to be largely scattered43, possibly
due to residual structure leading to varied compactness. In fact, thisinference is corroborated by detailed structural analyses for someof the 72 IDPs44–48 (Supplementary Note 5). Here we should emphasize that unlike SAXS, our HS-AFM method can single out IDRs in
the fully disordered state even when they undergo disorder–ordertransitions. We here selected ten tau-protein constructs that are notmuch affected by the extended repeat domains and phosphor-mimicmutation47. Then, we combined those 〈Rg〉 data with those of thefour PQBP-1 constructs herein reported. Fitting this data set to a
power law yielded 〈Rg〉 = (0.25 ± 0.038) × Naa0.53±0.028 (Supplementary
Fig. 6). A very close power law (〈Rg〉 = 0.254 × Naa0.522) was previously
obtained for several IDRs from their theoretical 〈Rg〉 values calculated using the coil database49. This agreement prompted us to use
these 14 experimental (Naa, 〈Rg〉) data to quantitate more accurately
the expansion effect of the mica surface on 〈R2D〉, while assumingthat this effect is independent of Naa. The measured 〈R2D〉 valuesof the IDRs (in the longer state when undergoing transitions)pwere
ﬃﬃﬃ
converted to corresponding 〈Rg〉 values as 〈Rg〉 = 〈R2D〉/(2 3u),where u is the expansion factor. Then, these nine (Naa, 〈Rg〉) dataI setscontaining the parameter u were combined with the above 14 SAXS
data sets of (Naa, 〈Rg〉). Finally, the combined data were fitted
to a power law, yielding u = 1.24 ± 0.01 and a fitted curve of〈Rg〉 = (0.26 ± 0.02) × Naa0.52±0.015, from which all data points only
slightly deviated (Extended Data Fig. 8a). From the 〈Rg(u)〉 values
at u = 1.24 for the nine IDRs, their Lp values in solution were calculated (Supplementary Table 8), yielding 〈Lp〉 = 0.78 ± 0.06 nm.
Similar Lp values can be found in various IDPs12,13,43 (Extended Data
Fig. 8b and Supplementary Table 9).

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

187

Articles

NATurE NAnoTEcHnology

Despite the rather small deviations of data points from the powerlaws of 〈Rg〉 = 0.26 × Naa0.52 and 〈R2D〉 = 1.16 × Naa0.52, it is advised to
avoid generalization of these power laws or the mean Lp values of
0.78 nm in solution and 1.18 nm on mica to all naturally occurring
IDRs in their fully disordered state. Indeed, they cannot be applied
to IDRs with a high charge density, a large fraction of charged residues (FCR) or largely segregated oppositely charged residues characterized by large values of κ (Supplementary Note 6 and Extended
Data Fig. 9). The global structure of IDRs has been indeed shown
to be modulated by electrostatic effects, that is a high charge density swells the volume of an IDR11,50, whereas a combination of large
FCR and large κ values results in protein compaction51,52. All IDPs
randomly chosen in this study do not possess high charge densities
or large values of FCR and κ (Supplementary Table 10); their charge
densities (ρ) range between –0.087 and +0.038. Small charge densities |ρ| ≤ 0.1 are often seen in numerous naturally occurring IDRs
(50.9% of all IDRs deposited in the Database of Protein Disorder,
Extended Data Fig. 9). Although the aa sequence and compositionof an IDR modulate its conformational sampling at various extents,
we resorted to use the power law and the mean Lp value on mica tomake a rough estimate of Naa encompassed by the fully disordered
IDRs contained in PNT and Sic1. The delineation from this Naa estimation of the global structure of PNT and its transitions appear to
be consistent with the domain diagram given by the recent NMR
analysis of PNT36 and with its proteolytic pattern37. In the case of
Sic1, the reliability of our quantitative estimation of its global structure and transitions cannot be assessed because structural information is currently limited. With the above-mentioned caveats, thepower laws and mean Lp values on mica and in solution obtained
above appear to be useful for semiquantitative delineation of thestructure and dynamics of IDPs, especially taking into account thecurrent paucity of more useful means for this analysis.

Conclusion

HS-AFM imaging of IDPs on mica can distinguish fully and partially disordered regions as well as fully ordered regions at thenear residue level, without fragmentation of the entire chain into
shorter pieces. Moreover, HS-AFM imaging can provide not only
length and height distributions for distinct regions in an IDP but
also information on the structural nature of the transiently folded
structures (for example, α-helix and flattened, collapsed structure)and their order propensity and transition dynamics. Thus, HS-AFM
imaging is an extremely powerful technique for characterizing
IDPs, in particular allowing resolving conformational heterogeneity
and avoiding the complications arising from ensemble averaging.
Thus, when all molecular features revealed by HS-AFM are combined with the folded local structure given by NMR, the combined
information allows a quantitative delineation of the structural anddynamic characters of IDPs, in a more realistic manner compared tothe pictures depicted individually, as demonstrated for PNT.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of
author contributions and competing interests; and statements ofdata and code availability are available at https://doi.org/10.1038/
s41565-020-00798-9.
Received: 9 January 2016; Accepted: 16 October 2020;
Published online: 23 November 2020

References

1. Romero, P. et al. Thousands of proteins likely to have long disordered regions.
Pac. Symp. Biocomp. 3, 437–448 (1998).
2. Uversky, V. N., Gillespie, J. R. & Fink, A. L. Why are ‘natively unfolded’ proteins
unstructured under physiologic conditions? Proteins 41, 415–427 (2000).

188

3. Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellularsignaling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015).
4. Dyson, H. J. & Wright, P. E. Unfolded proteins and protein folding studied by
NMR. Chem. Rev. 104, 3607–3622 (2004).
5. Jensen, M. R., Zweckstetter, M., Huang, J. & Blackledge, M. Exploring
free-energy landscapes of intrinsically disordered proteins at atomic
resolution using NMR spectroscopy. Chem. Rev. 114, 6632–6660 (2014).
6. Kikhney, A. G. & Svergun, D. I. A practical guide to small angle X-ray
scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett.
589, 2570–2577 (2015).
7. Dedmon, M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. &Dobson, C. M. Mapping long-range interactions in alphasynuclein using
spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem.
Soc. 127, 476–477 (2005).
8. Henriques, J., Cragnell, C. & Skepö, M. Molecular dynamics simulations of
intrinsically disordered proteins: force field evaluation and comparison withexperiment. J. Chem. Theory. Comput. 11, 3420–3431 (2015).
9. Bernadó, P. et al. A structural model for unfolded proteins from residual
dipolar couplings and small-angle X-ray scattering. Proc. Natl Acad. Sci. USA
102, 17002–17007 (2005).
10. Ozenne, V. et al. Flexible-meccano: a tool for the generation of explicit
ensemble descriptions of intrinsically disordered proteins and their associatedexperimental observables. Bioinformatics 28, 1463–1470 (2012).
11. Schuler, B., Soranno, A., Hofmann, H. & Nettels, D. Single-molecule FRET
spectroscopy and the polymer physics of unfolded and intrinsicallydisordered proteins. Annu. Rev. Biophys. 45, 207–231 (2016).
12. Borgia, A. et al. Consistent view of polypeptide chain expansion in chemical
denaturants from multiple experimental methods. J. Am. Chem. Soc. 138,
11714–11726 (2016).
13. Fuertes, G. et al. Decoupling of size and shape fluctuations inheteropolymeric sequences reconciles discrepancies in SAXS vs. FRET
measurements. Proc. Natl Acad. Sci. USA 114, E6342–E6351 (2017).
14. Ando, T. et al. A high-speed atomic force microscope for studying biologicalmacromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001).
15. Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force microscopy
for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83,
337–437 (2008).
16. Miyagi, A. et al. Visualization of intrinsically disordered regions of proteins by
high-speed atomic force microscopy. Chem. Phys. Chem. 9, 1859–1866 (2008).
17. Hashimoto, M. et al. Phosphorylation-coupled intramolecular dynamics of
unstructured regions in chromatin remodeler FACT. Biophys. J. 104,
2222–2234 (2013).
18. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force
microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333,
755–758 (2011).
19. Kodera, N., Yamamoto, T., Ishikawa, R. & Ando, T. Video imaging of walking
myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).
20. Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes byhigh-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014).
21. Ando, T. High-speed atomic force microscopy. Curr. Opin. Chem. Biol. 51,
105–112 (2019).
22. Takahashi, M. et al. Polyglutamine tract binding protein-1 is an intrinsically
unstructured protein. Biochim. Biophys. Acta 1794, 936–943 (2009).
23. de Gennes, P. –G. Scaling Concepts in Polymer Physics (Cornell Univ.
Press, 1979).
24. Frontali, C. Excluded-volume effect on the bidimensional conformation of
DNA molecules adsorbed to protein films. Biopolymers 27, 1329–1331 (1988).
25. Pérez, J., Vachette, P., Russo, D., Desmadril, M. & Durand, D. Heat-induced
unfolding of neocarzinostatin, a small all-beta protein investigated bysmall-angle X-ray scattering. J. Mol. Biol. 308, 721–743 (2001).
26. Iwao, T. Polymer Solutions: An Introduction to Physical Properties (John Wiley& Sons, Inc., 2002).
27. Kirk, J. & Ilg, P. Chain dynamics in polymer melts at flat surfaces.
Macromolecules 50, 3703–3718 (2017).
28. Yamamoto, H. et al. The intrinsically disordered protein Atg13 mediates
supramolecular assembly of autophagy initiation complexes. Dev. Cell 38,
86–99 (2016).
29. Mohan, A. et al. Analysis of molecular recognition features (MoRFs). J. Mol.
Biol. 362, 1043–1059 (2006).
30. Jensen, M. R. et al. Intrinsic disorder in measles virus nucleocapsids. Proc.
Natl Acad. Sci. USA 108, 9839–9844 (2011).
31. Gely, S. et al. Solution structure of the C‐terminal X domain of the measles
virus phosphoprotein and interaction with the intrinsically disordered C‐
terminal domain of the nucleoprotein. J. Mol. Recognit. 23, 435–447 (2010).
32. Oldfield, C. J. et al. Coupled folding and binding with α-helix-forming
molecular recognition elements. Biochemistry 44, 12454–12470 (2005).
33. Morin, B. et al. Assessing induced folding of an intrinsically disordered
protein by site-directed spin-labeling electron paramagnetic resonancespectroscopy. J. Phys. Chem. B. 110, 20596–20608 (2006).

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology
34. Belle, V. et al. Mapping α-helical induced folding within the intrinsicallydisordered C-terminal domain of the measles virus nucleoprotein bysite-directed spin-labeling EPR spectroscopy. Proteins 73, 973–988 (2008).
35. Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescentprotein. Science 273, 1392–1395 (1996).
36. Milles, S. et al. An ultraweak interaction in the intrinsically disorderedreplication machinery is essential for measles virus function. Sci. Adv. 4,
eaat7778 (2018).
37. Habchi, J., Mamelli, L., Darbon, H. & Longhi, S. Structural disorder within
henipavirus nucleoprotein and phosphoprotein: from predictions toexperimental assessment. PLoS ONE 5, e11684 (2010).
38. Brocca, S. et al. Compaction properties of an intrinsically disordered protein:Sic1 and its kinase-inhibitor domain. Biophys. J. 100, 2243–2252 (2011).
39. Riback, J. A. et al. Innovative scattering analysis shows that hydrophobic
disordered proteins are expanded in water. Science 358, 238–241 (2017).
40. Kohn, J. E. et al. Random-coil behavior and the dimensions of chemicallyunfolded proteins. Proc. Natl Acad. Sci. USA 101, 12491–12496 (2004).
41. Meier, S., Grzesiek, S. & Blackledge, M. Mapping the conformationallandscape of urea-denatured ubiquitin using residual dipolar couplings. J.
Am. Chem. Soc. 129, 9799–9807 (2007).
42. Le Guillo, J. C. & Zinn-Justin, J. Critical exponents for the n-vector model inthree dimensions from field theory. Phys. Rev. Lett. 39, 95–98 (1977).
43. Cordeiro, T. N. et al. Structural characterization of highly flexible proteins bysmall-angle scattering. Adv. Exp. Med. Biol. 1009, 107–129 (2017).
44. Boze, H. et al. Proline-rich salivary proteins have extended conformations.
Biophys. J. 99, 656–665 (2010).

45. Kate M. Nairn, K. M. et al. A synthetic resilin is largely unstructured.
Biophys. J. 95, 3358–3365 (2008).
46. Salmon, L. et al. NMR characterization of long-range order in intrinsicallydisordered proteins. J. Am. Chem. Soc. 132, 8407–8418 (2010).
47. Mylonas, E. et al. Domain conformation of tau protein studied by solution
small-angle X-ray scattering. Biochemistry 47, 10345–10353 (2008).
48. Lanza, D. C. et al. Human FEZ1 has characteristics of a natively unfolded
protein and dimerizes in solution. Proteins 74, 104–121 (2009).
49. Bernadó, P. & Blackledge, M. A. Self-consistent description of theconformational behavior of chemically denatured proteins from NMR andsmall angle scattering. Biophys. J. 97, 2839–2845 (2009).
50. Müller-Späth, S. et al. Charge interactions can dominate the dimensionsof intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 107,
14609–14614 (2010).
51. Das, R. K., Huang, Y., Phillips, A. H., Kriwacki, R. W. & Pappu, R. V. Cryptic
sequence features within the disordered protein p27Kip1 regulate cell cycle
signalling. Proc. Natl Acad. Sci. USA 113, 5616–5621 (2016).
52. Sherrya, K. P., Das, R. K., Pappu, R. V. & Barricka, D. Control oftranscriptional activity by design of charge patterning in the intrinsicallydisordered RAM region of the Notch receptor. Proc. Natl Acad. Sci. USA 114,
E9243–E9252 (2017).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2020

Nature Nanotechnology | VOL 16 | February 2021 | 181–189 | www.nature.com/naturenanotechnology

189

Articles
Methods

Preparation of PQBP-1 constructs. The DNA encoding full-length PQBP-1 wasinserted into a pGEX6P-1 plasmid that was used to transform the Escherichia coli
BL21 (DE3) strain. Protein expression was induced by the addition of 0.5 mM
isopropyl β-d-1-thiogalactopyranoside (IPTG). PQBP-1 was expressed as a
glutathione S-transferase (GST)-fusion protein. After cultivation, BL21 (DE3)cells were collected by centrifugation. The cells were resuspended in a buffer
containing 50 mM sodium phosphate (pH 7.0) and 2 mM dithiothreitol. After
cell lysis by sonication and removal of insoluble cell debris by centrifugation, thesupernatant was applied to a glutathione sepharose 4B column equilibrated with
50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol.
After washing the column with the same buffer, the GST fusion protein was eluted
from the column by adding 50 mM Tris-HCl (pH 8.5), 50 mM NaCl and 10 mM
reduced glutathione. To obtain PQBP-1, the GST fusion protein was cleaved witha recombinant rhinovirus 3C protease (PreScission protease, GE Healthcare). The
cleavage mixture was passed through the glutathione sepharose 4B column toremove GST and 3C protease. PQBP-1 with shorter IDRs and four IDR segments
were obtained in the same manner as the full-length PQBP-1. These constructs,
including WT PQBP-1, were finally purified by reverse phase high-performanceliquid chromatography using a 5C8-AR-300 column or a Protein-R column(Nacalai Tesque), followed by dialysis against 20 mM sodium phosphate (pH 7.0).
The molecular mass of PQBP-1 and its fragments was confirmed by matrix-assisted
laser desorption–ionization time-of-flight mass spectrometry.
Preparation of Atg1 and Atg13. Expression and purification of Atg1 (D211A)
were performed using the baculovirus expression system and tandem
chromatography as reported previously28. The DNA encoding C-terminal
His6-tagged full-length Atg13 was inserted into a pET11a plasmid (Novagen)that was used to transform the E. coli BL21 (DE3) strain. Protein expression was
induced by the addition of 0.1 mM IPTG. After cultivation, BL21 (DE3) cells werecollected by centrifugation. The cells were resuspended in a buffer containing
50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol,
5 mM 2-mercaptoethanol and protease inhibitor cocktail (Nacalai). After cell
lysis by sonication and removal of insoluble cell debris by centrifugation, thesupernatant was applied to a Ni-NTA column (Qiagen) equilibrated with 50 mM
Tris-HCl (pH 8.0), 500 mM NaCl and 10 mM imidazole. After washing the column
with the same buffer, the protein was eluted from the column by adding 50 mM
Tris-HCl (pH 8.0), 100 mM NaCl and 200 mM imidazole. Eluents were further
purified by gel filtration using a HiLoad 26/60 Superdex 200 PG column (GEHealthcare) and 20 mM Tris-HCl (pH 8.0), 150 mM NaCl as elution buffer. Purified
Atg1 (D211A) and Atg13 were concentrated using Centriprep (molecular cutoff of
50,000 Da) (Millipore) and then stored at −80 °C.
Preparation of GFP-fused proteins. The constructs encoding the GFP fusion
proteins used in this study have been already described53. They all encode forproteins consisting of an N-terminally hexahistidine tagged IDP (NTAIL, PNT or
Sic1), followed by a linker of 14 residues containing the tobacco etch virus proteasecleavage sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Ser) and the GFP, in thisorder (Fig. 4a–c). The E. coli strain Rosetta (DE3) pLysS (Novagen) was used forexpression of GFP fusions. Expression and purification were carried out as alreadydescribed53 except that immobilized metal affinity chromatography was performedby supplementing both equilibration and elution buffer with 1 M NaCl to get rid
of contaminating DNA. Eluents were further purified by gel filtration using a
Superdex 200 HR 16/60 column (GE Healthcare) and 50 mM sodium phosphate
pH 7.5, 200 mM NaCl as elution buffer. Proteins were concentrated using Centricon
Plus-20 (molecular cutoff of 10,000 Da) (Millipore) and then stored at −20 °C.
Preparation of Trx-fused NTAIL. The thioredoxin-6His-NTAIL (Trx-6His-NTAIL)
construct was generated as follows. The sequence encoding NTAIL followed by two
stop codons was subcloned by Gateway technology from an entry clone, whose
sequence had been checked by DNA sequencing54, into the Gateway expression
vector pETG-20A (a gift from A. Geerlof, EMBL) by LR reaction (a recombination
reaction between attL and attR sites). In this construct, the Trx-6His and NTAIL
moieties are separated by a linker (LYKKAGS) encoded by the Attb1 cloning siteborne by the pETG-20A vector. To obtain an NTAIL fusion protein only differing forthe position of the Trx tag, we designed a construct (referred to as NTAIL-6His-Trx)bearing the same Attb1-encoded sequence between the 6His tag and Trx. Since
another Attb1 cloning site is also present in the cloning vector (pDEST14, see
below), the Attb1 sequence added between the 6His tag and Trx was mutated (initalics in the primer sequences given below) in such a way that it could not berecognized by the LR enzyme while still encoding the same linker as WT attb1.
The coding sequence of NTAIL was PCR amplified using the same entry clone
bearing the NTAIL coding sequence as above54, and primers Attb1-SD-NT(GTACAAAAAAGCAGGCTAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCGACTACTgaggacaagatcag)
and NT-link(GTCATGGTACCATGATGATGATGATGGTGCATATGGCCAGAACCAGAACCGTCTAGAAGATTTCTGTCATTG). The coding sequence of
Trx was PCR amplified using pETG-20A as template and primers link-TRX

NATurE NAnoTEcHnology(CCATCATCATCATCATGGTACCATGAAGccTGTAtAAgAAg
GCgGGtagcAGCGATAAAATTATTCACC)
and TRX-stop-Attb2(ACCACTTTGTACAAGAAAGCTGGGTttattaGGCCAGGTTAGCGTCGAGGAAC).
After DpnI digestion, 1 µl of each of the first two PCR product were mixed andthen used as template in a third PCR amplification with primers attL1a(ACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGC
CAAGTTTGTACAAAAAAGCAGGC)
and attL2a(TTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCCACTTTGTACAAGAAAGCTGG)55. The third PCRamplification allows to (1) fuse the first two PCR products, (2) insert a 6His tag(underlined in the above primer sequences) and the Attb1-encoded linker between
NTAIL and Trx and (3) add Attl1a and Attl2a sequences, respectively, at the 5′ and 3′
end of the PCR product required for the subsequent cloning step by LR reaction.
The product of the third PCR was then inserted into the Gateway vector pDEST14
by LR reaction and checked by DNA sequencing. A detailed description of the two
fusion proteins is provided in Supplementary Fig. 1.
The E. coli strain T7pRos54 was used for expression of Trx-NTAIL and NTAIL-Trx
fusion proteins. Cultures were grown overnight to saturation in LB medium
containing 100 µg ml−1 ampicillin. An aliquot of the overnight culture was diluted
1:25 in LB medium and grown at 37 °C. At an optical density (OD600) of 0.7, IPTG
was added to a final concentration of 0.5 mM, and the cells were grown at 37 °C
for 3 h. The induced cells were collected by centrifugation (6,000g for 20 min at 4 °C)
and resuspended in 30 ml of buffer A (50 mM Tris-HCl, pH 8, 500 mM NaCl, 20 mM
Imidazole, 1 mM PMSF), supplemented with 0.1 mg ml−1 of lysozyme, 10 µg ml−1 of
DNase I and protease inhibitor cocktail (Roche). After a 20-min incubation withgentle agitation, the cells were disrupted by sonication (using a 750 W sonicator andthree cycles of 30 s each at 45% power output). Following the removal of cell debris by
centrifugation at 14,000g for 30 min, the supernatant was applied onto a Ni(II)-loaded
5-ml His-Trap high-performance column (GE Healthcare) previously equilibrated
in buffer A. The affinity resin was washed with 20 column volumes of buffer A
containing 1 M NaCl. After this step, the affinity column was connected in tandem
to a Superdex 75 16/60 column (GE Healthcare) pre-equilibrated in NaP 10 mM,
NaCl 200 mM pH 7 buffer. The two connected columns were eluted first using 20 mlof buffer A containing 500 mM imidazole and subsequently using 10 mM phosphatebuffer at pH 7, containing 200 mM NaCl. The fractions containing the recombinant
protein were collected, lyophilized and stored at −20 °C. For both proteins, mass
spectrometry analysis yielded a value that fits perfectly with the expected molecularmass for a form in which the initial methionine has been cleaved off.
SAXS analysis. SAXS measurements were performed at 23 °C with a BioSAXS-1000
system (Rigaku) mounted on a MicroMx007HF X-ray generator (Rigaku). A
PILATUS100K detector (DECTRIS) at a distance of 462 mm from the sample,
was used to measure scattering intensities. The IDR segments of PQBP-1 (82–134,
82–164, 82–214 and 82–265) in 10 mM phosphate buffer (pH 6.0) were used for
SAXS measurements. Circular averaging of the scattering intensities was carriedout to obtain one-dimensional scattering data I(q) as a function of q (q = 4π sinθ/λ,where 2θ is the scattering angle and λ is the X-ray wavelength 1.5418 Å) by using
the program SAXSlab (Rigaku). The I(q) data were processed by using the software
applications embedded in the ATSAS package56. The radius of gyration Rg andforward scattering intensity I(0) were estimated from the Guinier plot of I(q) in
the smaller angle region of qRg < 1.3. The molar mass of the IDR segments were
estimated by comparing I(0)/c (where c is the protein concentration) of the IDR
segments to that of ovalbumin, or estimated from QR (ref. 57). To correct interparticle
interference, I(q) data were collected at three different protein concentrations (10, 8
and 6 mg ml−1 for 82–134 and 82–164; 5, 3 and 0.3 mg ml−1 for 82–214; 14, 10 and
8 mg ml−1 for 82–265). Because the intensity profile of 82–134, 82–164 and 82–265
did not indicate a concentration effect, the correction for interparticle interference
was not applied. The 82–214 fragment exhibited weak self-association at 5 and
3 mg ml−1. Therefore, a diluted sample (0.3 mg ml−1) was used for structural analysis.
SAXS data are summarized in Extended Data Fig. 2 and Supplementary Table 3.
HS-AFM observation. The procedure for HS-AFM observation of protein
dynamics at the single-molecule level has already been described58. In brief, aglass sample stage (diameter, 2 mm; height, 2 mm) with a thin mica disc (1 mmin diameter and 0.05 mm thick) glued to the top by epoxy was attached onto
the top of the Z scanner by a drop of nail polish. A freshly cleaved mica surface
was prepared by removing the top layers of mica using Scotch tape. Then a drop
(2 μl) of the diluted sample (around 3 nM) was deposited onto the mica surface.
After an incubation of about 3 min, the mica surface was rinsed with 20 μl of anobservation buffer shown below for each sample to remove floating samples. The
sample stage was then immersed in a liquid cell containing around 60 μl of thesame observation buffer. HS-AFM observation was performed in the tapping modeusing a laboratory built apparatus14,15. The short cantilevers used are custom-madeby Olympus; resonant frequency, around 1 MHz in water; quality factor, about 2 inwater; spring constant, 0.1–0.15 N m−1. The cantilever’s free oscillation amplitude
A0 and set point amplitude As were set at 1–2 nm and around 0.9 × A0, respectively,
Nature Nanotechnology | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology
so that the loss of cantilever’s oscillation energy per tap was adjusted to be 1–3
kBT on average. The imaging rate, scan size and the pixel size for each sample
are described in the main text and Supplementary Information. The observationbuffers used for the respective IDPs are as follows:
PQBP-1

10 mM sodium phosphate buffer, pH 6.0

Atg1

20 mM KCl and 20 mM Tris-HCl, pH 6.0
20 mM KCl and 20 mM Tris-HCl, pH 7.0
20 mM KCl and 20 mM Tris-HCl, pH 8.0
50 mM NaCl and 20 mM Tris-HCl, pH 8.0
300 mM NaCl and 20 mM Tris-HCl, pH 8.0

Atg13

100 mM NaCl and 20 mM Tris-HCl, pH 6.0
100 mM NaCl and 20 mM Tris-HCl, pH 7.0
100 mM NaCl and 20 mM Tris-HCl, pH 8.0

FACT-10SA

50 mM KCl, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.5 and
0.5% glycerol (vol/vol)NTAIL-GFP

10 mM sodium phosphate buffer, pH 6.0

NTAIL-Trx

10 mM sodium phosphate buffer, pH 6.0
10 mM sodium phosphate buffer, pH 7.0

Trx- NTAIL

10 mM sodium phosphate buffer, pH 6.0

PNT–GFP

10 mM sodium phosphate buffer, pH 6.0

Sic1-GFP

10 mM sodium phosphate buffer, pH 6.0

Analysis of AFM images. To measure topographical parameters (R2D, H1 and H2),
we used a pixel search software program that we had made for a previous HS-AFM
study59. This program is opened to the public at the following web site: https://
elifesciences.org/content/4/e04806/article-data#fig-data-supplementary-material.
First, AFM images are briefly pretreated (mostly flattened for scan line
non-horizontality) by our AFM-specific data acquisition software. From an AFM
image, the operator roughly finds an end region of IDR and then clicks the mousepointer at a molecule-free position on the surface that appears very close to theend region. Then, the pixel search program automatically finds a pixel position(x, y) having the largest height Z, by searching over n × n pixels around theoperator-specified pixel position (the value of n is appropriately chosen, typicallyn = 5). The height H (H1 or H2) is obtained by subtracting the average height of thesubstrate surface from the z value. The same procedure is repeated at the other endof the IDR. From the obtained two sets of (x, y) coordinates, the direct distance D
between the two pixels is calculated, which is followed by a calculation of R2D valueas R2D = D − H1/2 − H2/2. The autocorrelation functions G(τ) of time-series datax(nΔt) of R2D and H2 were calculated as
XN
XN�hGðτÞ 
½xðnΔt Þ � ^μ½xððn þ hÞΔt Þ � ^μ=½xðnΔt Þ � ^
μ2 ;
n¼1
n¼1

References

53. Sambi, I., Gatti–Lafranconi, P., Longhi, S. & Lotti, M. How disorderinfluences order and vice versa – mutual effects in fusion proteins
containing an intrinsically disordered and a globular protein. FEBS J. 277,
4438–4451 (2010).
54. Gruet, A., Longhi, S. & Bignon, C. One-step generation of errorprone PCR libraries using Gateway® technology. Microb Cell Fact. 11,
15 (2012).
55. Gruet, A. et al. Fuzzy regions in an intrinsically disordered protein impair
protein-protein interactions. FEBS J. 283, 576–594 (2016).
56. Petoukhov, M. V. et al. New developments in the ATSAS program
package for small-angle scattering data analysis. J. Appl. Cryst. 45,
342–350 (2012).
57. Rambo, R. P., John, A. & Tainer, J. A. Accurate assessment of
mass, models and resolution by small-angle scattering. Nature 496,
477–481 (2013).
58. Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure
dynamics and dynamic processes of proteins by high-speed atomic force
microscopy. Nat. Protoc. 7, 1193–1206 (2012).
59. Ngo, K. X., Kodera, N., Katayama, E., Ando, T. & Uyeda, T. Q. P.
Cofilin-induced unidirectional cooperative conformational changes in actin
filaments revealed by high-speed AFM. eLife 4, e04806 (2015).

Acknowledgements

We thank M. Hakozaki (Kanazawa University) for her technical assistance, H.
Okazawa (Tokyo Medical and Dental University) for providing the human PQBP-1
complementary DNA and A. Schramn (Laboratory AFMB UMR 7257, Aix-Marseille
University and CNRS) for providing the statistics of IDRs deposited in the DisProt
database. This work was supported by grants from the MEXT, Japan, Grants-in-Aid
for Scientific Research on Innovative Areas: Research in a Proposed Research Area,
grant nos. 21113002 and 26119003 (T.A.), no. 21113003 (M.M.), nos. 15H01651 and
17H05894 (Y.F.) and nos. 25111004 and 19H05707 (N.N.N.), Grant-in-Aid for Basic
Research S, grant nos. 24227005 and 17H06121 (T.A.), CREST program of JST, grant no.
JPMJCR13M1 (T.A.), and no. JPMJCR13M7 (N.N.N.), PRESTO program of JST (N.K.),
and Grant-in-Aid for Young Scientists, grant no. 19K16344 (D.N.), and partly supported
by grants from the Agence Nationale de la Recherche, specific programs ‘Microbiologie
et Immunologie’ ANR-05-MIIM-035-02 (S.L.) and ‘Physico-Chimie du Vivant’ ANR08-PCVI-0020-01 (S.L.). D.B. was supported by a joint doctoral fellowship from the
Direction Générale de l’Armement and the CNRS. E.S. was supported by a joint doctoral
fellowship from the Direction Générale de l’Armement and Aix-Marseille University.
M.D. was supported by a PhD fellowship from the French Ministry of National
Education, Research and Technology.

Author contributions

Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.

S.L. and T.A. designed the project. M.L. provided the original constructs encoding GFP
fusions. M.M., D.B., J.H., A.G., M.D., E.S., C.B., Y.F. and N.N.N. prepared constructs
and/or protein samples used in this study. T.A. and N.K. developed the HS-AFM system.
N.K., D.N., S.K.D. and T.M. performed the HS-AFM experiments. N.K., T.A., D.N.,
S.K.D. and T.M. analysed HS-AFM data. T.O. and M.S. performed the SAXS experiments
and analysed SAXS data. T.A. made all theoretical formulations and wrote the draftof the manuscript. S.L., N.K. and T.A. prepared the final manuscript based on thediscussions performed among all authors.

Data availability

Competing interests

where τ ≡ hΔt and ^μ is the mean value of x(nΔt) (n = 1, 2, 3,..., N).

All data that support the findings of this study have been included in the main
text, Extended Data figures and Supplementary Information. The original source
data associated with all figures are attached to respective figures. Source data areprovided with this paper.

Code availability

The original code for AFM image analyses is opened to the public at the following
web site: https://elifesciences.org/content/4/e04806/article-data#fig-datasupplementary-material

Nature Nanotechnology | www.nature.com/naturenanotechnology

The authors declare no competing interests.

Additional information

Extended data is available for this paper at https://doi.org/10.1038/s41565-020-00798-9.
Supplementary information is available for this paper at https://doi.org/10.1038/
s41565-020-00798-9.
Correspondence and requests for materials should be addressed to S.L. or T.A.
Reprints and permissions information is available at www.nature.com/reprints.

Articles

NATurE NAnoTEcHnology

Extended Data Fig. 1 | Molecular features of PQBP-1 (1−214) observed at various imaging rates. a−f, H1, H2 and R2D distributions measured from HS-AFM
images captured at 6.7 (a), 10.0 (b), 15.2 (c), 20.0 (d), 30.3 (e) and 50.0 (f) fps. The most probable fitting curves are drawn with the solid lines. g, 〈H1〉 atvarious imaging rates. h, 〈H2〉 at various imaging rates. i, 〈R2D〉 at various imaging rates. Each plot corresponds to mean values ± s.e.m. measured at eachframe rate. The horizontal lines indicate the weighted mean values. Details of these analyses are summarized in Supplementary Table 2. Note that theintermittent tip-sample contact force becomes larger with increasing imaging rate, whereas the number of contacts per frame increases with decreasing
imaging rate. Nevertheless, the values of 〈R2D〉, 〈H1〉 and 〈H2〉 are nearly constant, irrespective of the imaging rate, indicating no notable impact of the
tip-sample contact on the structure of this protein.

Nature Nanotechnology | www.nature.com/naturenanotechnology

NATurE NAnoTEcHnology

ArticlesExtended Data Fig. 2 | I(q) curves, Guinier plots, and Kratky plots obtained from SAXS measurements of IDR segments of PQBP-1 (82−134,
82−164, 82−214 and 82−265). a−d, I(q) curves displayed in a q range from 0.018 to 0.500 Å-1. e−h, Guinier plots displayed in the smaller regionof qRg < 1.3. i, Kratky plots normalized by I(0) indicating the fully disordered nature of the four IDRs. A summary of these analyses is presented in
Supplementary Table 3.

Nature Nanotechnology | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology

Extended Data Fig. 3 | Characterization of Atg1 (D211A) and R2D distributions of its IDR measured under various solution conditions. a, Domain diagramof the Atg1 (D211A) construct (light blue, IDR) and its order/disorder map along its length as predicted by DISPROT (VSL2). b, Coomassie Brilliant
Blue-stained SDS-PAGE (10%) of WT Atg1 and Atg1 (D211A) showing autophosphorylation of WT Atg1 and no phosphorylation of Atg1 (D211A). The
autophosphorylation reaction was performed by incubating 2 μM Atg1 with 2 mM ATP and 5 mM MgCl2 in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl buffer.
The dephosphorylation reaction was performed by incubating 2 μM Atg1 with 1 μM lambda protein phosphatase (λPPase), 2 mM dithiothreitol and 1 mM
MnCl2 in the same buffer. These reactions were terminated by the addition of Laemmli SDS sample buffer. c-f, R2D distributions of IDR measured undervarious solution conditions. The most probable fitting curves are shown with the solid blue lines. The black lines represent the Gaussian components indouble-Gaussian fitting. Note that the 〈R2D〉 values at the second peaks (longer states) are nearly identical irrespective of the solution condition, whereas the〈R2D〉 values at the first peaks (shorter states) vary depending on the solution condition. A summary of these analyses is presented in Supplementary Table 4.

Nature Nanotechnology | www.nature.com/naturenanotechnology

NATurE NAnoTEcHnology

ArticlesExtended Data Fig. 4 | Order/disorder map along the length of Atg13 and R2D distributions of its IDR measured at two pH values. a, Domain diagramof the Atg13 construct used in this study and order/disorder map along its length as predicted by DISPROT (VSL2). The regions indicated by the red bars
are 359–389, 424–436 and 641-661 (Atg17-binding regions) and 460–521 (Atg1-binding region). b, c, R2D distributions of IDR measured at pH 6.0 (b) andpH 8.0 (c). The most probable fitting curves are shown with the solid blue lines. The black lines represent the Gaussian components in double-Gaussian
fitting. Note that the 〈R2D〉 values at the second peaks (longer states) are nearly identical irrespective of the solution condition, whereas the 〈R2D〉 values at
the first peaks (shorter states) slightly vary depending on pH. A summary of these analyses is presented in Supplementary Table 5.

Nature Nanotechnology | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology

Extended Data Fig. 5 | Height of fully disordered IDRs contained in three IDP-GFP fusions. To make sure that the IDRs under analysis are fully disordered,
HS-AFM images showing longer IDRs were chosen. a, NTAIL-GFP. b, PNT-GFP. c, Sic1-GFP. d, Height of IDR in Sic1-GFP as a function of its distance from the
N-terminus. AFM images of Sic1-GFP molecules showing IDR longer than 25 nm were chosen in this height analysis to ensure that the IDR under analysis
was formed upon order-to-disorder transition of the N-terminal small globule. The height of IDR was measured as a function of the distance from the
N-terminal globule with a bin width of 2.5 nm. The height values for different frames and molecules measured within the bin width at each lateral distancewere averaged and the mean height was plotted together with s.e.m. The mean height of the N-terminal end (0.8 nm) is slightly smaller than the height ofpeak 1 (0.9 nm) shown in Fig. 4m in the main text. This is due to the relatively large bin width (2.5 nm) of the lateral distance used in this analysis.

Nature Nanotechnology | www.nature.com/naturenanotechnology

NATurE NAnoTEcHnology

ArticlesExtended Data Fig. 6 | Structural features of Trx-NTAIL observed by HS-AFM imaging. a, Domain diagram of Trx-NTAIL (the a.a. sequence is given in
Supplementary Fig. 1). Color codes: Grey, Trx; orange, linkers; white, His6; red, α-More; cyan, IDR. b, Successive HS-AFM images of Trx-NTAIL observed at pH
6.0. The observed molecular features are schematized just above the respective AFM images. The closed and open arrow heads point at the Trx moiety
and the tail end, respectively. c, Height distribution of the Trx domain. d, R2D distribution of the IDR. e, Height distribution of the C-terminal globule (Box2).
The most probable fitting curves are shown with the thick solid lines. The thin black lines in (e) indicate two Gaussian components in double-Gaussian
fitting. A summary of these analyses is presented in Supplementary Table 7.

Nature Nanotechnology | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology

Extended Data Fig. 7 | Structural features of NTAIL-Trx observed by HS-AFM imaging. a, Domain diagram of NTAIL-Trx (the a.a. sequence is given in
Supplementary Fig. 1). Color code: black, Met and Ser; the others are the same as in Extended Data Fig. 6. b, c, Successive HS-AFM images of NTAIL-Trx
molecules observed at pH 6.0 (b) and pH 7.0 (c). The closed and open arrow heads point at the Trx moiety and the tail end, respectively. d, g, Height
distributions of the Trx domain measured at pH 6.0 (d) and pH 7.0 (g). e, h, R2D distributions measured at pH 6.0 (e) and pH 7.0 (h). f, i, Height
distributions of the N-terminal small globule (Box1) measured at pH 6.0 (f) and pH 7.0 (i). The most probable fitting curves are shown with thicksolid lines (d−i). The thin black lines in (f, i) are two Gaussian components in double-Gaussian fitting. A summary of these analyses is presented in
Supplementary Table 7.

Nature Nanotechnology | www.nature.com/naturenanotechnology

NATurE NAnoTEcHnology

ArticlesExtended Data Fig. 8 | Refinement of expansion effect of mica on the 2D dimensions of IDR and power law for 〈Rg〉. a, 〈Rg〉−Naa relationship for 23 data.
The 10 data plotted with the red circles are those from the study by Mylonas et al. for tau protein constructs47, with phosphor-mimic constructs and thoselargely affected by the extended repeat domain43,49 having been removed here. The constructs herein analyzed are tau ht40, tau K32, tau K16, tau ht23,
tau K27, tau K17, tau K44, tau K10, tau K25, and tau K23. The data plotted with the blue circles are those from the present SAXSpstudy
for four segments
ﬃﬃﬃ
of PQBP-1 IDR (82−265, 82−214, 82−164, and 82−134). The nine data plotted with the green circles are those of (Naa, 〈R2D〉/(2 3u) at u = 1.24) of the
I used). The value of u
five PQBP-1 constructs, Atg1, Atg13, FACT protein and NTAIL (〈R2D〉 data highlighted with orange color in Supplementary Table 8 are
were determined by fitting all 23 data to a power law, 〈Rg〉 = βg × Naaν, in which the parameter u was contained in the nine data as one of the variables tobe determined. The black solid line indicates the best fitting result; u = 1.24, βg = 0.26 nm, and ν = 0.52. b, 〈Rg〉−Naa relationship for 37 data. The 37 data
points include 14 data found from literature search (black circles; Supplementary Table 9) and those 23 data shown in (a). Fitting of the 37 data points to a
power law, 〈Rg〉 = βg × Naaν, yielded βg = 0.260 ± 0.021 nm and ν = 0.524 ± 0.015 (black line).

Nature Nanotechnology | www.nature.com/naturenanotechnology

Articles

NATurE NAnoTEcHnology

Extended Data Fig. 9 | Statistics of charge-associated parameters for many naturally occurring IDRs. a, 2D plot of (Naa, ρ) (ρ, charge density) of naturally
occurring IDRs whose information is deposited in the Database of Protein Disorder that gathers information of IDRs that have been confirmed to bedisordered. The paired values (Naa, ρ) of 1,011 IDRs are plotted in this graph. Half of these data points (50.9%) are within the range of |ρ| ≤ 0.1. Note thatshorter IDRs are enriched in the database, reflecting the fact that short IDPs tend to be more experimentally characterized than longer IDRs. b, Histogramof κ for the IDRs contained in the database; IDRs with Naa ≤ 19 were omitted. Each bar of the histogram is divided into four regions according to the value offraction of charged residues (FCR). The CIDER software program (opened to public use) was used to calculate the values of κ and FCR (see Supplementary
Note 6). c, Scatter diagram of (FCR, κ) for the IDRs contained in the database; IDRs with Naa ≤ 19 were omitted. The points shown in red (4.5%)
correspond to IDRs that meet the combined condition of κ ≥ 0.35 & FCR ≥ 0.3 that is considered to be required for the appearance of a distinct volume
compaction effect of oppositely charged residues52. It would be plausible that a volume compaction effect could be manifest even for IDRs possessing a
FCR value less than (for example) 0.25, when they have a very large value of κ. To depict such a possibility, the red line is drawn (κ × FCR = 0.35 × 0.30).
It should be kept in mind however that this is just tentative and awaits future experimental confirmation.

Nature Nanotechnology | www.nature.com/naturenanotechnology

Last updated by author(s): Oct 9, 2020

Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency
in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist .

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

nature research | reporting summary

Corresponding author(s): Toshio Ando, Sonia Longhi

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement
A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient)AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated
Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code
Data collection

Provide a description of all commercial, open source and custom code used to collect the data in this study, specifying the version used OR
state that no software was used.

Data analysis

HS-AFM images were first treated with the lab-made software Kodec for noise reduction and flattening. The length and height analyses of
HS-AFM images were made using the lab-made pixel-search software program. Histograms were analyzed using Origin. The autocorrelation functions of time-series of height data were calculated using Mathematica v.10.

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers.
We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

The data that support the protein sample preparations and all graphs within this paper are available from the corresponding authors upon request. The code of the
pixel-search software program used for AFM image analysis can be accessed athttps://elifesciences.org/content/4/e04806/article-data#fig-data-supplementary-material
All custom code used in this study is available upon request.

October 2018

- Accession codes, unique identifiers, or web links for publicly available datasets- A list of figures that have associated raw data
- A description of any restrictions on data availability

1