Image analysis

All lines scans were flattened with a linear fit to the background of each line, similar to previous AFM image analysis (36,48). Images were analysed using a semi-automated algorithm (39). Binding of PRC2 was associated with a distinct increase in measured height along the axis of DNA. Occasionally, DNA adopted a configuration where the helical backbone crossed itself in the absence of protein. The frequency of such looped structures was ∼8-fold lower when using our deposition protocol that yielded equilibrated DNA with a more extended molecular configuration than standard deposition protocols that yielded kinetically trapped DNA configurations when imaging in liquid (39). Notwithstanding this lower frequency of false positives, we acquired negative controls (i.e. images of DNA without protein). When higher-resolution images were acquired so an individual molecule almost filled the full range of the image (typically 600 × 600 nm²), looped structures were distinguishable from protein bound to the DNA both by height and a volumetric analysis (see section: monomers and multimers of PRC2 distinguished).

Volumetric analysis is the standard analysis for determing the molecular weight and/or the multimeric state of complexes (46,49–51), with an understanding that the resulting deduced volumes inherently include a convolution of the AFM tip radius. As detailed in Supplementary Figure S7, volume analysis was performed by first bounding the region of interest (DNA loop or protein-DNA complex), then fitting a 2D, freely rotating, elliptical Gaussian function to the height as a function of x and y. The volume was measured by integrating the Gaussian over the region of interest. To improve the volume estimation due to inter-image variability in AFM cantilever radius, the DNA in an image was used as a volumetric calibration for that image, conceptually similar to prior work that used naked DNA as a volume standard (52). In our implementation, the DNA height perpendicular to the DNA contour was measured at least 50 nm in contour length from loops or protein. Fitting and then integrating a 1D Gaussian to each height profile along a DNA contour length yielded a DNA volume per unit contour length (Supplementary Figure S7D, E). On a per-molecule basis, the volume of the DNA loop or DNA–PRC2 complex was multiplied by the ratio of the expected to the measured DNA volume per contour length. For simplicity, we assumed the DNA volume per contour length was πr² based on a rod with a radius of 1 nm. Effectively, this procedure yields the volume of the DNA loop or PRC2 complex, where the volume for each molecule is scaled based on the expected volume per unit contour length of the DNA. We note that omitting our deconvolution procedure did not substantially change the overall shape of the volume distribution or interpretation of the data (Supplementary Figure S8).

To demonstrate that the DNA molecules were equilibrated in 2D on the mica surface, we quantified the DNA’s persistence length (p). A DNA molecule was defined as equilibrated if analysis of its 2D conformation with a 2D WLC model yielded the correct value of p, a definition consistent with prior DNA imaging studies (36,39,53). To do so, we measured the angle θ between two tangent vectors separated by arc length s, a standard analysis (36,53). For this analysis, we selected interpretable DNA molecules, defined as molecules with a configuration containing two or fewer strand crossings. The tangent vector was determined by fitting a third-order, least-squared polynomial spline through user-defined points spaced ∼10 nm along the DNA molecules, excluding looped segments. We then fit the resulting data to

which is appropriate for analyzing a molecule in 2D (53) (Supplementary Figure S9). This analysis yielded p = 49.1 ± 0.4 nm (mean ± std. dev.; N = 640) consistent with DNA’s known persistence length of 50 nm (54).