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. 2023 Nov 3;14:7072. doi: 10.1038/s41467-023-42641-4

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Illustration of the DNA configuration found in the PFV strand transfer complex (STC) crystal structure (PDB 3OS0) showing the fluorophore positions on the vDNAs (Cy3-PFV) and the target DNA (F-Cy5). The estimated inter-dye distances, corresponding FRET efficiencies, and the average FRET value (E_STC) of a mixture of molecules containing a single Cy3 on the left or right vDNA. b, c Representative intensity trajectories and corresponding FRET trajectories with HMM fits showing stable strand transfer by a Cy3-PFV intasome. Green arrows mark the photobleaching of the Cy3 FRET donor. The Post-synchronized histogram and smFRET histograms corresponding to STC events for target DNAs containing a 1nt Gap (5’-P) (d) or 1nt Gap (5’-OH) (e). The Gaussian fits to the FRET histograms are shown as red lines. Data were collected at 100 ms frame rate (b) or 1 s frame rate (c–e). Inset shows the total number of transitions with >0.1 FRET (n) and the percentage (%) of strand transfer events. The post-synchronized histogram and smFRET histograms corresponding to TCC (f) and STC (g) events for Cy3-PFV-ddA interacting with 1nt Gap (5’-OH) target DNA. Data were collected at 100 ms frame rate (f) and 1 s frame rate (g). Inset shows the total number of DNA molecules analyzed (N) (f); and the number of stable transitions with >0.1 FRET (n) and the percentage (%) of STC events (g; N = 564). h Illustration of the DNA configuration found in the PFV strand transfer complex (STC) crystal structure (PDB 3OS0) showing the fluorophore positions on the vDNAs (Cy3) and the reverse-Cy5 (R-Cy5) target DNA, respectively. The estimated inter-dye distances, corresponding FRET efficiencies, and the average FRET value (E_TCC) of a mixture of molecules containing a single Cy3 on the left or right vDNA. i A representative intensity trajectory (top) and the corresponding FRET trajectory (bottom) with the HMM fit showing Cy3-PFV binding to R-Cy5 target DNA containing a 1nt Gap (5’-OH). j The post-synchronized histogram (left) and the smFRET histogram (right) produced by averaging the total number (N) of TCC smFRET traces. k A representative intensity trajectory (top) and the corresponding FRET trajectory (bottom) with HMM fit showing Cy3-PFV strand transfer into R-Cy5 target DNA containing a 1nt Gap (5’-OH). I The post-synchronized histogram (left) and the smFRET histogram (right) corresponding to the total number (n) and percentage (%) of STC events. The data in (i–l) were collected at 1 s frame rate.