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. 2018 Jan 16;9:235. doi: 10.1038/s41467-017-02619-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Multiscale chromatin dynamics in two registers revealed by MFD. a Scheme of PIE-MFD: Species-averaged donor and acceptor emission intensities (F_D, F_A), intensity-averaged donor lifetime 〈τ_D(A)〉_F and anisotropy (r) are simultaneously measured for each molecule diffusing through the confocal volume. b Principle of MFD analysis: If dynamics between two states A and D are slow (relaxation time t_R ≫ 10 ms), distinct structural states are resolved by E_FRET and 〈τ_D(A)〉_F, falling on the static FRET line (red). c Fast dynamics (t_R < 10 ms) result in an intermediate peak (labeled A ↔ D) on a dynamic FRET line (blue). Peak shape analysis reveals t_R (Fig. 5). d 2D-MFD histograms for chromatin fibers DA1–3 (Alexa568/647) at indicated Mg²⁺ concentrations. These histograms contain contributions from donor-only labeled chromatin fibers. Red line: static FRET line. Dark and bright blue lines: Two dynamic FRET lines for the two tetranucleosome registers 1 and 2, indicating dynamic exchange with t_R < 10 ms (for parameters of all FRET lines, see Supplementary Note, step 2). Red, orange, yellow, and gray lines: FRET species A–D (see also Fig. 4a). e 2D-MFD histograms for chromatin fibers DA1–3 labeled with Alexa488/647 at indicated Mg²⁺ concentrations. Red line: static FRET line; dark and bright blue lines: dynamic FRET lines. f Subensemble fluorescence lifetime analysis for DA1-labeled fibers (Alexa488/647) at 1 mM MgCl₂ and E_FRET>0.065. The FRET-induced donor decay ε_D(t) was fitted with contributions from FRET species {A, C}, B and D (for details and fit parameters of eqs. 3.1–6, see Supplementary Note, step 3), corresponding to the indicated inter-dye distances. IRF: instrument response function. g Auto- (left panel) and cross (right panel)-correlation functions of the donor (G) and acceptor (R) emission channels for the same subensemble as in f. Global analysis of FCS curves reveals FRET dynamics with two global structural relaxation times (t_R2 = 27 µs (27%); t_R3 = 3.1 ms (56 %)), a term describing local fluctuations (t_R1,local = 2.6 µs (17%)) and an apparent diffusion time for all curves (t_diff = 4.96 ms) (for details and fit parameters of Eq. 4.1, see Supplementary Note, step 4)