Fig. 3: Spatial distribution, stoichiometry, and diffusive behavior of loading intermediates observed in ATPγS.

a Histograms of the spatial distribution of fluorescent foci after a 2-min incubation in the flow cell. (i) Stacked bins representing foci containing 1 or 2 ORC (green) or colocalized ORC and MCM (orange), and (ii) foci containing 1 or 2 MCM (red) molecules. The overall stoichiometry distributions of these foci are shown in panel (iii). The gray bin labeled “other” accounts for all colocalized foci where either the ORC or MCM stoichiometry is 3 or higher. b As in a, but following an incubation time of 8 min. c As in a and b, but following a 30-min incubation in bulk, ×1000 dilution, and introduction into the flow cell. d Sample time traces and corresponding image to illustrate the motion of colocalized JF549-ORC (green) and JF646-MCM (red) acquired at a frame rate of 0.6 s. e–g Histograms of the diffusion constants of the loading intermediates shown in a–c. The populations of diffusion constants fit log-normal distributions (solid black lines), taking into account the error bars derived from bootstrapping the data set 100 times. e After a 2-min incubation in the flow cell, the fitted diffusion coefficients are (mean ± SEM, in units of kbp² s⁻¹): ORC: 0.02 ± 0.04 and 1.0 ± 0.4 (p = 0.008 by one-way ANOVA); O-M: 0.07 ± 0.06; MCM: 0.009 ± 0.005 and 1.0 ± 0.3 (p = 0.04 by one-way ANOVA). f After an 8 min incubation, the diffusion coefficients are (mean ± SEM, in kbp² s⁻¹): ORC: 0.01 ± 0.02 and 0.28 ± 0.09 (p = 0.007 by one-way ANOVA); O-M: 0.012 ± 0.007 and 0.39 ± 0.09 (p < 0.0001 by one-way ANOVA); MCM: 0.9 ± 2.3. g After the bulk 30-min incubation and introduction into the flow cell, the diffusion coefficients are (mean ± SEM, in kbp² s⁻¹): ORC: 0.008 ± 0.009; O-M: 0.006 ± 0.003; MCM: 0.04 ± 0.06. h Summary of the fitted diffusion constants in e–g for different incubation times and conditions (e.g., in-flow-cell or bulk).