Abstract
Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural 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 folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
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 are provided 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-data-supplementary-material
References
Romero, P. et al. Thousands of proteins likely to have long disordered regions. Pac. Symp. Biocomp. 3, 437–448 (1998).
Uversky, V. N., Gillespie, J. R. & Fink, A. L. Why are ‘natively unfolded’ proteins unstructured under physiologic conditions? Proteins 41, 415–427 (2000).
Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellular signaling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015).
Dyson, H. J. & Wright, P. E. Unfolded proteins and protein folding studied by NMR. Chem. Rev. 104, 3607–3622 (2004).
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).
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).
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).
Henriques, J., Cragnell, C. & Skepö, M. Molecular dynamics simulations of intrinsically disordered proteins: force field evaluation and comparison with experiment. J. Chem. Theory. Comput. 11, 3420–3431 (2015).
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).
Ozenne, V. et al. Flexible-meccano: a tool for the generation of explicit ensemble descriptions of intrinsically disordered proteins and their associated experimental observables. Bioinformatics 28, 1463–1470 (2012).
Schuler, B., Soranno, A., Hofmann, H. & Nettels, D. Single-molecule FRET spectroscopy and the polymer physics of unfolded and intrinsically disordered proteins. Annu. Rev. Biophys. 45, 207–231 (2016).
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).
Fuertes, G. et al. Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements. Proc. Natl Acad. Sci. USA 114, E6342–E6351 (2017).
Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001).
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).
Miyagi, A. et al. Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy. Chem. Phys. Chem. 9, 1859–1866 (2008).
Hashimoto, M. et al. Phosphorylation-coupled intramolecular dynamics of unstructured regions in chromatin remodeler FACT. Biophys. J. 104, 2222–2234 (2013).
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).
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).
Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014).
Ando, T. High-speed atomic force microscopy. Curr. Opin. Chem. Biol. 51, 105–112 (2019).
Takahashi, M. et al. Polyglutamine tract binding protein-1 is an intrinsically unstructured protein. Biochim. Biophys. Acta 1794, 936–943 (2009).
de Gennes, P. –G. Scaling Concepts in Polymer Physics (Cornell Univ. Press, 1979).
Frontali, C. Excluded-volume effect on the bidimensional conformation of DNA molecules adsorbed to protein films. Biopolymers 27, 1329–1331 (1988).
Pérez, J., Vachette, P., Russo, D., Desmadril, M. & Durand, D. Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering. J. Mol. Biol. 308, 721–743 (2001).
Iwao, T. Polymer Solutions: An Introduction to Physical Properties (John Wiley & Sons, Inc., 2002).
Kirk, J. & Ilg, P. Chain dynamics in polymer melts at flat surfaces. Macromolecules 50, 3703–3718 (2017).
Yamamoto, H. et al. The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes. Dev. Cell 38, 86–99 (2016).
Mohan, A. et al. Analysis of molecular recognition features (MoRFs). J. Mol. Biol. 362, 1043–1059 (2006).
Jensen, M. R. et al. Intrinsic disorder in measles virus nucleocapsids. Proc. Natl Acad. Sci. USA 108, 9839–9844 (2011).
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).
Oldfield, C. J. et al. Coupled folding and binding with α-helix-forming molecular recognition elements. Biochemistry 44, 12454–12470 (2005).
Morin, B. et al. Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy. J. Phys. Chem. B. 110, 20596–20608 (2006).
Belle, V. et al. Mapping α-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy. Proteins 73, 973–988 (2008).
Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Milles, S. et al. An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function. Sci. Adv. 4, eaat7778 (2018).
Habchi, J., Mamelli, L., Darbon, H. & Longhi, S. Structural disorder within henipavirus nucleoprotein and phosphoprotein: from predictions to experimental assessment. PLoS ONE 5, e11684 (2010).
Brocca, S. et al. Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain. Biophys. J. 100, 2243–2252 (2011).
Riback, J. A. et al. Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water. Science 358, 238–241 (2017).
Kohn, J. E. et al. Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl Acad. Sci. USA 101, 12491–12496 (2004).
Meier, S., Grzesiek, S. & Blackledge, M. Mapping the conformational landscape of urea-denatured ubiquitin using residual dipolar couplings. J. Am. Chem. Soc. 129, 9799–9807 (2007).
Le Guillo, J. C. & Zinn-Justin, J. Critical exponents for the n-vector model in three dimensions from field theory. Phys. Rev. Lett. 39, 95–98 (1977).
Cordeiro, T. N. et al. Structural characterization of highly flexible proteins by small-angle scattering. Adv. Exp. Med. Biol. 1009, 107–129 (2017).
Boze, H. et al. Proline-rich salivary proteins have extended conformations. Biophys. J. 99, 656–665 (2010).
Kate M. Nairn, K. M. et al. A synthetic resilin is largely unstructured. Biophys. J. 95, 3358–3365 (2008).
Salmon, L. et al. NMR characterization of long-range order in intrinsically disordered proteins. J. Am. Chem. Soc. 132, 8407–8418 (2010).
Mylonas, E. et al. Domain conformation of tau protein studied by solution small-angle X-ray scattering. Biochemistry 47, 10345–10353 (2008).
Lanza, D. C. et al. Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution. Proteins 74, 104–121 (2009).
Bernadó, P. & Blackledge, M. A. Self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering. Biophys. J. 97, 2839–2845 (2009).
Müller-Späth, S. et al. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 107, 14609–14614 (2010).
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).
Sherrya, K. P., Das, R. K., Pappu, R. V. & Barricka, D. Control of transcriptional activity by design of charge patterning in the intrinsically disordered RAM region of the Notch receptor. Proc. Natl Acad. Sci. USA 114, E9243–E9252 (2017).
Sambi, I., Gatti–Lafranconi, P., Longhi, S. & Lotti, M. How disorder influences order and vice versa – mutual effects in fusion proteins containing an intrinsically disordered and a globular protein. FEBS J. 277, 4438–4451 (2010).
Gruet, A., Longhi, S. & Bignon, C. One-step generation of error-prone PCR libraries using Gateway® technology. Microb Cell Fact. 11, 15 (2012).
Gruet, A. et al. Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions. FEBS J. 283, 576–594 (2016).
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).
Rambo, R. P., John, A. & Tainer, J. A. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496, 477–481 (2013).
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).
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’ ANR-08-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 information
Authors and Affiliations
Contributions
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 draft of the manuscript. S.L., N.K. and T.A. prepared the final manuscript based on the discussions performed among all authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
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〉 at various 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 each frame rate. The horizontal lines indicate the weighted mean values. Details of these analyses are summarized in Supplementary Table 2. Note that the intermittent 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.
Extended 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 region of 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.
Extended Data Fig. 3 Characterization of Atg1 (D211A) and R2D distributions of its IDR measured under various solution conditions.
a, Domain diagram of 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 under various solution conditions. 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) vary depending on the solution condition. A summary of these analyses is presented in Supplementary Table 4.
Extended Data Fig. 4 Order/disorder map along the length of Atg13 and R2D distributions of its IDR measured at two pH values.
a, Domain diagram of 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) and pH 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.
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 distance were 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 of peak 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.
Extended 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.
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 thick solid 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.
Extended 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 those largely 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 SAXS study for four segments 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\(\sqrt 3 u\)) at u = 1.24) of the five PQBP-1 constructs, Atg1, Atg13, FACT protein and NTAIL (〈R2D〉 data highlighted with orange color in Supplementary Table 8 are used). The value of u 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 to be 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).
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 be disordered. 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 that shorter IDRs are enriched in the database, reflecting the fact that short IDPs tend to be more experimentally characterized than longer IDRs. b, Histogram of 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 of fraction 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.
Supplementary information
Supplementary Information
Supplementary Notes 1–6, Figs. 1–6 and Tables 1–10.
Supplementary Video 1
HS-AFM video of WT PQBP-1. Molecules of WT PQBP-1 were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 23.8 fps for a scan area of 80 × 80 nm2 with 80 × 80 pixels. This video is played back at 24 fps.
Supplementary Video 2
HS-AFM video of Atg1 (D211A variant). Molecules of Atg1 (D211A) were placed on a mica surface in 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl and imaged at 10 fps for a scan area of 100 × 100 nm2 with 80 × 80 pixels. This video is played back in real time.
Supplementary Video 3
HS-AFM video of Atg13. Molecules of Atg13 were placed on a mica surface in 20 mM Tris-HCl (pH 7.0) containing 100 mM NaCl and imaged at 10 fps for a scan area of 120 × 120 nm2 with 100 × 100 pixels. This video is played back in real time.
Supplementary Video 4
HS-AFM video of NTAIL-GFP. Molecules of NTAIL-GFP were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 10 fps for a scan area of 80 × 100 nm2 with 80 × 80 pixels. This video is played back in real time.
Supplementary Video 5
HS-AFM video of Trx-NTAIL. Molecules of Trx-NTAIL were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 10 fps for a scan area of 60 × 60 nm2 with 80 × 80 pixels. This video is played back in real time.
Supplementary Video 6
HS-AFM video of NTAIL-Trx. Molecules of NTAIL-Trx were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 10 fps for a scan area of 60 × 60 nm2 with 80 × 80 pixels. This video is played back in real time.
Supplementary Video 7
HS-AFM video of PNT–GFP. Molecules of PNT–GFP were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 10 fps for a scan area of 80 × 100 nm2 with 100 × 100 pixels. This video is played back in real time.
Supplementary Video 8
HS-AFM video of Sic1-GFP. Molecules of Sic1-GFP were placed on a mica surface in 10 mM phosphate buffer (pH 6.0) and imaged at 10 fps for a scan area of 80 × 100 nm2 with 80 × 80 pixels. This video is played back in real time.
Supplementary Data
Source Data for Supplementary Figs. 2–6.
Source data
Source Data Fig. 1
Statistical Source Data
Source Data Fig. 2
Statistical Source Data
Source Data Fig. 3
Statistical Source Data
Source Data Fig. 4
Statistical Source Data
Source Data Extended Data Fig. 1
Statistical Source Data
Source Data Extended Data Fig. 2
Statistical Source Data
Source Data Extended Data Fig. 3
Statistical Source Data
Source Data Extended Data Fig. 4
Statistical Source Data
Source Data Extended Data Fig. 5
Statistical Source Data
Source Data Extended Data Fig. 6
Statistical Source Data
Source Data Extended Data Fig. 7
Statistical Source Data
Source Data Extended Data Fig. 8
Statistical Source Data
Source Data Extended Data Fig. 9
Statistical Source Data
Rights and permissions
About this article
Cite this article
Kodera, N., Noshiro, D., Dora, S.K. et al. Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy. Nat. Nanotechnol. 16, 181–189 (2021). https://doi.org/10.1038/s41565-020-00798-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41565-020-00798-9
This article is cited by
-
Intrinsically disordered regions in TRPV2 mediate protein-protein interactions
Communications Biology (2023)
-
End-to-end differentiable blind tip reconstruction for noisy atomic force microscopy images
Scientific Reports (2023)
-
Visualizing single-molecule conformational transition and binding dynamics of intrinsically disordered proteins
Nature Communications (2023)
-
Dimerization processes for light-regulated transcription factor Photozipper visualized by high-speed atomic force microscopy
Scientific Reports (2022)
-
An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution
Scientific Reports (2021)
