In every time-lapse FRET trace of Rb-WT (ribosomes bearing wild type L27), the sampling frequencies of the three FRET states (Hy1, C, and Hy2) are different, indicating the existence of multiple ribosome subpopulations with distinct local dynamics. To characterize the uniqueness of each ribosome, the FRET traces are analyzed by FRET efficiency histograms to determine the normalized composition of the FRET states. Figure 2(a) displays one example of the original fluorescence intensities from both the donor and the acceptor. The calculated FRET efficiencies are shown in Figure 2(b). Figure 2(c) displays the Gaussian fittings of the FRET efficiency histogram. The amount of each FRET state is calculated as the area underneath the Gaussian fitting curve. The composition of the Hy1, C, and Hy2 states are calculated to be 55%, 27.4%, and 17.6%, respectively (the fitting details are described in the Supporting Information and Figure S4). In this ribosome, all three FRET states are sampled. Figure 2(d–f) display another ribosome that only samples the C and Hy2 states (0%, 58.2%, and 41.8% for Hy1, C, and Hy2, respectively). More examples are shown in Supporting Information Figure S5. Therefore, every ribosome can be identified by its signature of FRET state composition. The determinations of these signatures are statistically reliable because of the sufficient data points for each ribosome.
Figure 2. Calculation of the FRET states compositions. (a) One example of a pair of donor-acceptor fluorescence intensities. The presence of a single ribosome is confirmed by single step bleaching. (b) The FRET efficiency based on (a) is calculated as efficiency = Iacceptor/(Iacceptor + Idonor). (c) Histogram analysis of the FRET efficiencies in (b). Regardless of the limited data points (on average 110 points before bleaching), the Gaussian fittings are reasonable. (d), (e), and (f) provide another single ribosome example that are similar to (a), (b), and (c). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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For the Rb-WT, we studied 3695 particles of pretranslocation complexes and plotted them on a three-dimensional (3D) graph [Fig. 3(a)]. The X-, Y-, and Z-axes are the compositions of the Hy1, C, and Hy2 states, respectively. All of the ribosomes are located in the plane of X + Y + Z = 1 because the compositions are normalized to 1. The ribosomes that are located at (1,0,0), (0,1,0), and (0,0,1) are the static subpopulations that only sample one FRET state. For example, ribosomes at (1,0,0) sample 100% of the Hy1 state. Ribosomes at (0,0,1) sample 100% of the P/P state (the Hy2 state only exists in fluctuating ribosomes as discussed above). The ribosomes that lie in the XY, XZ, or YZ planes only sample two of the FRET states. For example, ribosomes in the XY plane only sample the Hy1 and C states, and so on. Ribosomes that sample all three FRET states do not intercept the axis planes. For example, the ribosome trace shown in Figure 2(a) locates at position (X = 0.55, Y = 0.274, Z = 0.176) in Figure 3(a), which means it samples the Hy1, C, and Hy2 state 55%, 27.4%, and 17.6%, respectively. Accordingly, as shown in Figure 3(b), we sort the subpopulations into seven categories based on the ribosomes' locations on the 3D coordinates and calculate the percentages of each subpopulation. Previously, we have sorted the ribosomes into nonfluctuating, fluctuating without the Hy2 state, and fluctuating with the Hy2 state categories based only on their FRET values.8 Here, by assigning a unique 3D identifier to each ribosome, seven subpopulations are visible: ∼ 20% of the Hy1-only and C-only states (nonfluctuating), 25% of the Hy1-C state (fluctuating without the Hy2 state), 5% of the P/P state (spontaneous translocation), and 30% of the rest (fluctuating with the Hy2 state). These results agree well with our previous sorting results.
Figure 3. The 3D-plot and subpopulation sorting of the WT ribosome pre-translocation complexes. (a) The 3D-plot of the ribosomes according to their FRET state percentages: X, Y, and Z-axes are the percentages for the Hy1, C, and Hy2 states, respectively. Plot (a) sorts the ribosome into seven subpopulations according to the location of the data points. (b) Normalized ribosome subpopulation distributions for plot (a). The name for each subpopulation is the same as in (a). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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