Extended Data Fig. 8: Single-molecule experimentation and analysis.
From: Architecture of an HIV-1 reverse transcriptase initiation complex
a, Secondary structure depiction of the vRNA–tRNA construct used for single-molecule experiments. The labelling scheme is shown, with the Cy3 dye located on the 5′ end of the vRNA helix 1 and Cy5 dye located on an oligonucleotide positioned near the 5′ end of helix 1. The vRNA–tRNA complex was crosslinked to RT for the experiments. b, Ninety-five per cent of the RTIC complexes are in the high FRET, helix 1 formation, state (480 traces analysed, see Methods). c, Example trace of the ones used for final FRET analysis. The high FRET state of the RTIC complex, which is attributed to helix 1 formation. Photobleaching events for both Cy5 and Cy3 are indicated. d, Examples of traces removed from final FRET analysis. Traces exhibit the presence of multiple molecules (multiple single-dye photobleaching events) or poor dye behaviour (blinking and quenching).
