Nature Structural & Molecular Biology: doi: /nsmb.3175

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2 Supplementary Figure 1 Medium- and low-fret groups probably representing varying positions on the DNA show similar changes between H3 and CENP-A nucleosomes with and without CENP-C CD, as does the high-fret state. Summary of FRET values and changes for nucleosomes and DNA indicated for medium and low FRET groups. The (a) medium and (d) low FRET values for H3, CENP-A, and CENP-A nucleosomes bound by CENP-C CD were plotted as histograms and fitted to a Gaussian distribution for DNA1 and DNA2. Efficiency values for (b) medium and (e) low are the center of Gaussian curve fitting and the errors are the uncertainty at a 95% c.i. The differences in FRET for (c) medium and (f) low FRET groups are calculated for three different nucleosome comparisons (indicated in columns A and B) on both DNA1 and DNA2. While the predominant species of nucleosomes are positioned with the central bp of the 147 bp sequence located at the dyad, there can be subpopulations of nucleosomes with the dyad shifted by one or a few bp in either direction. The nature of single-molecule experiments allows for measurement of these subpopulations, which are reflected in the medium and low FRET groups. Both of these groups follow the same FRET trends as the high FRET group, but represent a smaller percentage of the total measurements. Thus, we interpret them as nucleosomes that have a different dyad position than the predominant high FRET group. Medium FRET; DNA1: N = 129 (H3), 103 (CENP-A), 226 (CENP-A + CENP-C CD ); DNA2: N = 143 (H3), 209 (CENP-A), 102 (CENP-A + CENP-C CD ). Low FRET; DNA1: N = 68 (H3), 44 (CENP-A), 35 (CENP-A + CENP-C CD ); DNA2: N = 60 (H3), 58 (CENP-A), 25 (CENP-A + CENP-C CD ). N represents the total number of individual nucleosome measurements taken from 3 separate slides.

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4 Supplementary Figure 2 The four-helix bundle of the heterotetramer exhibits differential rotation and compaction in the CENP-A H4 tetramer, CENP-A nucleosome and H3 nucleosome crystal structures. To illustrate the rotation at the four-helix bundle near the dyad, the 2 helix (residues ) of CENP-A from the CENP-A nucleosome crystal structure (PDB ID 3AN2; pink) and the (CENP-A H4) 2 heterotetramer (PDB ID 3NQJ; red) were overlaid on the 2 helix (residues ) of H3 from a high resolution nucleosome structure (PDB ID 1KX5; green). All three structures show a different rotation at the four-helix bundle, most evidently seen when comparing the orientations of the 2' helices. The 2 helices in the H3 nucleosome crystal structure form the widest angle. Relative to the H3 H3 orientation, there is an ~8 rotation in the CENP-A nucleosome crystal structure and an ~12 rotation in the (CENP-A H4) 2 heterotetramer crystal structure, suggesting that both CENP-A complexes exhibit rotation at the four-helix bundle interface. The rotation in the structure of the (CENP-A H4) 2 heterotetramer is accompanied by compaction (inward movements of the heterodimers), shortening the distance between the N-terminal ends of the 2 helices. Thus, the CENP-A nucleosome crystal structure has a CENP-A CENP-A interface that exhibits rotation but not compaction, representing an intermediate structure between the (CENP-A H4) 2 heterotetramer and the H3 nucleosome crystal structures.

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6 Supplementary Figure 3 ENA footprinting of nucleosomes reveals stretching at a contact point with CENP-A H4. (a) Map of the 5 -fluorescently labeled top strand of the 147-bp -satellite DNA sequence used in ENA footprinting experiments. Basepair numbering corresponds to the 5 (-) to 3 (+) direction relative to the dyad. The GG dinucleotide discussed in (c) is boxed. (b) Native PAGE analysis of H3, CENP-A, and CENP-A + CENP-C CD nucleosomes complexed with (+) or without (-) ENA. Top, ethidum bromide staining, bottom, coomassie staining. The free DNA template is overrepresented in the ethidium bromide staining because nucleosomes restrict its intercalation. (c) Representative image of ENA footprinting results (N=3 independent experiments). Samples were run on a denaturing PAGE gel against a Maxam-Gilbert purine standard (marker) that allows for single basepair resolution of relevant regions of the nucleosome. ENA selectively alkylates guanines, making them prone to thermally induced chemical cleavage and allowing for footprinting analysis. In all cases, the nucleosome core from the dyad to +40 is protected from ENA intercalation relative to free DNA. The -14:-15 GG dinucleotide, which has been previously shown to be the major site of increased ENA incorporation in the stretched 145 bp canonical nucleosome (in the context of a palindromic DNA template with the same sequence as the negative half of our native -satellite template) (Davey, GE. et al., Nucleic Acids Res. 38, , 2010), shows differential cleavage patterns in H3, CENP-A, and CENP-A + CENP-C CD nucleosomes. There is a marked increase in cleavage at this site in the CENP-A nucleosome compared to the H3 nucleosome, while the effect in the CENP-A + CENP-C CD nucleosome is intermediate between the two. This is likely due in part to the rotation in the CENP-A nucleosome relative to the H3 nucleosome and the differing local protein DNA environment between CENP-A and H3 nucleosomes in this region of the nucleosome. We conclude that the important DNA stretching in the CENP-A nucleosome occurs along the surface wrapping the (CENP-A H4) 2 heterotetramer. Finally, the DNA termini show a similar trend of ENA accessibility as the -14:-15 GG dinucleotide, with an intermediate result for CENP-A + CENP- C CD nucleosomes. While there are no GG dinucleotides to facilitate ENA intercalation, the more flexible termini (relative to the dyad) allow for easier ENA alkylation at G mononucleotides. We previously reported that CENP-C CD binding alters the DNA termini of the CENP-A nucleosome (Falk, SJ. et al., Science. 348, , 2015), so we interpret these changes in ENA footprinting to indicate that when CENP-C CD binds, the histone core interactions with the DNA are altered to a structured and rigid state (i.e. less ENA reactivity) but where the DNA is more easily digested with nuclease and DNA dimensions are increased as measured by SANS with contrast variation (Falk, SJ. et al., Science. 348, , 2015). Colored boxes correspond to the respective line scans for each sample, which highlight nucleosome protection, and the cleavage patterns at the -14:-15 GG dinucleotide and DNA termini.

7 H3 nucleosomes CENP-A nucleosomes CENP-A + CENP-C CD nucleosomes DNA1 DNA2 DNA1 DNA2 DNA1 DNA2 Low FRET Medium FRET High FRET Total Supplementary Table 1. Summary of the different Φ FRET groups observed in smfret measurements. The number and percentage of the nucleosomes analyzed in this study binned into the low, medium, and high Φ FRET groups (Fig. 1 and Supplementary Fig. 1).