Figure S5.
Linkage error investigation and number of localizations per clathrin coat. (A–L) Impact of linkage error on geometric model fit. Indirect immunolabeling displaces the label from the target molecule by two antibodies. This generates a so-called linkage error of on average ± 10 nm, and resulting localizations might not accurately represent the underlying structure of interest (Früh et al., 2021). (A) An ideal case of no linkage error, where the model (dotted black line) with radius RM accurately represents the underlying clathrin coat (red) with a radius RC. (B) Histogram of quantified curvature with a median of 0.011 nm−1. (C) Models fitted to curvature propagation over θ. (D) Uniform displacement of localizations (light red, ± 10 nm) due to unbiased labeling by the antibodies. The radius RM still accurately represents the true radius RC. (E) Histogram of the curvature, assuming no needed correction of RM. Median: 0.011 nm−1. (F) Models fitted to curvature propagation over θ remain the same as for C. (G) Biased labeling of the antibodies (light red, + 10 nm) could result in an overestimation of RM by 10 nm. (H) Histogram of the curvature corrected by subtracting 10 nm from quantified RM. Median: 0.013 nm−1. (I) Models fitted to corrected curvature propagation over θ. (J) Biased labeling could result in an underestimation of RM by 10 nm. (K) Histogram of the curvature corrected for the overestimation in radius by adding 10 nm from quantified RM. Median: 0.010 nm−1. (L) Models fitted to corrected curvature propagation over θ. (C, F, I, and L) Fitting parameters are A0: Fraction of surface area growing as a flat lattice before curvature initiation, defined as A0 = A(θ = 0.01)/A(θ = π); R0: Preferred radius of the clathrin coat fitted with CoopCM; H0: Preferred curvature of the clathrin coat fitted with CoopCM. Analysis of N = 13 SK-MEL-2 cells, n = 1,645 sites. While the fitting parameters scale with the error in radius estimation, the relationships among the parameters and thus our mechanistic interpretation by the cooperative curvature model still holds true. (M–O) Number of localizations versus surface area. For N = 6 SK-MEL-2 cells, n = 700 sites the number of localizations found in one clathrin-coated structure was extracted. This is plotted against the quantified surface area determined for each coat.

Linkage error investigation and number of localizations per clathrin coat . (A–L) Impact of linkage error on geometric model fit. Indirect immunolabeling displaces the label from the target molecule by two antibodies. This generates a so-called linkage error of on average ± 10 nm, and resulting localizations might not accurately represent the underlying structure of interest (Früh et al., 2021). (A) An ideal case of no linkage error, where the model (dotted black line) with radius RM accurately represents the underlying clathrin coat (red) with a radius RC. (B) Histogram of quantified curvature with a median of 0.011 nm−1. (C) Models fitted to curvature propagation over θ. (D) Uniform displacement of localizations (light red, ± 10 nm) due to unbiased labeling by the antibodies. The radius RM still accurately represents the true radius RC. (E) Histogram of the curvature, assuming no needed correction of RM. Median: 0.011 nm−1. (F) Models fitted to curvature propagation over θ remain the same as for C. (G) Biased labeling of the antibodies (light red, + 10 nm) could result in an overestimation of RM by 10 nm. (H) Histogram of the curvature corrected by subtracting 10 nm from quantified RM. Median: 0.013 nm−1. (I) Models fitted to corrected curvature propagation over θ. (J) Biased labeling could result in an underestimation of RM by 10 nm. (K) Histogram of the curvature corrected for the overestimation in radius by adding 10 nm from quantified RM. Median: 0.010 nm−1. (L) Models fitted to corrected curvature propagation over θ. (C, F, I, and L) Fitting parameters are A0: Fraction of surface area growing as a flat lattice before curvature initiation, defined as A0 = A(θ = 0.01)/A(θ = π); R0: Preferred radius of the clathrin coat fitted with CoopCM; H0: Preferred curvature of the clathrin coat fitted with CoopCM. Analysis of N = 13 SK-MEL-2 cells, n = 1,645 sites. While the fitting parameters scale with the error in radius estimation, the relationships among the parameters and thus our mechanistic interpretation by the cooperative curvature model still holds true. (M–O) Number of localizations versus surface area. For N = 6 SK-MEL-2 cells, n = 700 sites the number of localizations found in one clathrin-coated structure was extracted. This is plotted against the quantified surface area determined for each coat.

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