Figure 1. High resolution structure of SNF2h bound to a nucleosome with 60 bp of flanking DNA in the presence of ADP-BeF_x and 140 mM KCl.

(A) Cryo-EM density map of SNF2h bound to the nucleosome at 3.4 Å from data recorded with a K2-summit camera. (B) Model built using the density in (A). (C) Cartoon representation of a nucleosome with asymmetric flanking DNA as in our structures. Super Helical Location (SHL) ± 2 as well as the entry and exit site DNAs are labeled. The SHL0 location is also labeled and is defined as the dyad. Faces A and B of the histone octamer are labeled in gray. (D) Zoom into the ATP-binding pocket of SNF2h with ADP in orange and represented with sticks. In spheres are the SNF2h residues that bind nucleotide with the helicase motif I in green and helicase motif VI in blue (Figure 3—figure supplement 4).

Figure 1—figure supplement 1. Cryo-EM analysis of singly bound SNF2h-nucleosome complexes (140 mM KCl) .

(A) A raw cryo-EM micrograph of single bound SNF2h-nucleosome complex recorded as described in Materials and methods. Examples of monodisperse particles are circled. (B) Slices through the unsharpened density of the 3.9 Å resolution map, which has SNF2h bound to the SHL-2 position, at different levels along the top view. (C) Typical 2D class averages of selected particles. The box size is 256 pixels. (D) Representative views of the 3D reconstruction of the 3.4 Å resolution . (E) Representative views of the 3.9 Å resolution map with SNF2h bound to the SHL-2 position. (F) Representative views of the 6.9 Å resolution map with SNF2h bound to the SHL+2 position. (G) Euler angle distribution of all particles used for the final 3D reconstruction of the 3.4 Å resolution. The length of each cylinder is proportional to the number of particles visualized from this specific orientation. (H) Euler angle distribution of all particles used for the 3.9 Å 3D reconstruction. The length of each cylinder is proportional to the number of particles visualized from this specific orientation. (I) Euler angle distribution of all particles used for the 6.9 Å resolution 3D reconstruction. (J) FSC curves of the 3.4 Å complex between two independently refined half maps before (red) and after (blue) masking in Cryosparc v2, indicated with resolutions corresponding to FSC = 0.143. (K) Final unsharpened 3.4 Å 3D density map of SNF2h-nucleosome complex colored with local resolution. (L) FSC curves of the 3.9 Å reconstruction between two independently refined half maps before (red) and after (blue) masking in Cryosparc, indicated with resolutions corresponding to FSC = 0.143. (M) FSC curves of the 6.9 Å complex (N) Cross-validation using FSC curves of the density map calculated from the refined model versus half map 1 (‘work’, blue), versus half map 2 (‘free’, pink) and versus summed map (green).

Figure 1—figure supplement 2. Cryo-EM Densities of SHL+2 and SHL-2 SNF2h-Nucleosome complexes obtained at 140 mM.

KCl Cryo-EM reconstructions of single-SNF2h bound nucleosomes from data recorded on a K2-summit direct electron bound at (A) SHL +2 at 6.9 Å resolution and (B) SHL-2 at 3.9 Å resolution.

Figure 1—figure supplement 3. Cryo-EM reconstructions of the SNF2h-Nucleosome complexes at 140 mM KCl are translocated ~2 bp.

(A) High resolution structure from Figure 1—figure supplement 2B with Cryo-EM density fit with an atomic model. The additional 2 bp of DNA exiting the canonical nucleosome structure are highlighted in red. The box indicates the region zoomed to show greater detail in (B). (B) Zooms of the boxed region in A to show greater detail. The phosphates of the phosphodiester backbone are shown in spheres and fit precisely into the Cryo-EM density. (C) Schematic of the ensemble FRET experiment performed in (D). Nucleosomes were reconstituted on identical DNA sequence used to obtain Cryo-EM reconstructions and labeled with two fluorophores: cy3 on the 5’ phosphate at the DNA exit side of the nucleosome with a three carbon flexible linker and with cy5 on C8 of the cytosine base at position 149 near the entry side of the nucleosome via a six carbon flexible linker. Upon addition of SNF2h and ADP-BeF_x, if the nucleosome is translocated as in the Cryo-EM reconstruction, the two labeled bases will be brought closer together in 3D space. This change in DNA conformation could be read out as a very small increase in FRET efficiency between the two labels. (D) Ensemble FRET experiment described in (C). FRET efficiency values were normalized to the FRET efficiency of the nucleosome prior to adding SNF2h and nucleotide. Points represent the mean of the indicated number independent experiments and error bars represent the standard error of the mean. Addition of SNF2h and ADP-BeF_x produces an instantaneous drop in FRET efficiency that slowly increases in the presence 140 mM KCl but not in the presence of 70 mM KCl (top). The instantaneous drop in FRET efficiency may be due to subtle changes in the conformation of DNA immediately upon SNF2h binding or due to previously reported changes in the photophysics of the cy3 dye labeled at this position (Racki et al., 2009). The subsequent increase in FRET reflects structural changes in nucleosomal DNA which, in principle, could reflect many types of motions such as subtle changes in DNA trajectory. However, the increase observed is consistent with the small ~2 bp translocation captured in our cryo-EM reconstructions. Changes in the photophysical properties of the cy5 label were ruled out as contributing to the change in FRET efficiency as there was no change in fluorescence intensity when cy5 was directly excited (data not shown). No increase in FRET signal was seen when only SNF2h buffer was added (bottom).

Figure 1—figure supplement 4. By a single molecule assay, SNF2h induces a change in FRET under the 140 mM KCl conditions, consistent with a movement of the nucleosomal DNA.

(A) Setup of the single molecule assay. Nucleosomes with 78 bp flanking DNA on one side are attached to the surface of a microscope slide and imaged using total internal reflection fluorescence microscopy. The nucleosomes have 9 bp flanking DNA on the other side, with a Cy5 dye at the end of the 9 bp. Movement of the DNA will change the distance between the Cy3 and Cy5 dyes, changing the observed FRET. (B) Kernel density estimation (KDE) plot of the starting FRET values of 179 nucleosomes, without SNF2h, combined from the three independent replicates shown in (C). FRET values cluster into two peaks, near 0.8 FRET and 0.45 FRET, corresponding to two possible labeling schemes: a Cy3 dye on the histone H3 closer to the Cy5-labeled DNA end (proximal labeling), or a Cy3 dye on the other copy of histone H3 (distal labeling). As demonstrated in Zhou et al. (2018), the distally-labeled population is relatively insensitive to changes in the length of the DNA separating the Cy5 from the edge of the nucleosome, and so is not shown in (D) and (E). (C) KDEs (left) and empirical cumulative distribution functions (CDFs; right) of starting nucleosomal FRET values from three independent replicates. Note that the FRET value of the midpoint of a peak in a KDE corresponds to the FRET value where the slope of the CDF is steepest. Thus the distal and proximal peaks in the KDEs appear as two regions of steep slopes in the CDFs. For two data sets with similar peak locations, the FRET value (position along the x-axis) where the slope of the CDF is steepest will be similar. (D) (black) KDE of FRET values for 43 proximally-labeled nucleosomes after 10 min of incubation without SNF2h, and (red) 48 proximally-labeled nucleosomes after a 10 min incubation in the presence of 2 µM SNF2h and 0.5 mM ADP-BeF_x. Dashed lines labeled ‘+11’, ‘+10’, and ‘+9’ indicate expected FRET values for nucleosomes with 11, 10, or 9 bp of flanking DNA between the Cy5 and the edge of the nucleosome, based on the calibration curve derived in Zhou et al. (2018). (E) Empirical cumulative distribution functions (CDFs) of the data in B and C. (F) Same as (C) but for the two replicates of nucleosomes alone after a 10 min incubation. The two replicates have 115 and 117 nucleosomes each (with the distally labeled population included). (G) Same as (C) but for the two replicates of nucleosomes plus SNF2h and ADP-BeF_x after 10 min. The two replicates have 72 and 83 nucleosomes each. Shaded regions in CDFs in all panels represent an estimate of the error based on a bootstrapping method (Gamarra et al., 2018; Zhou et al., 2018).

Figure 1—figure supplement 5. Difference maps to test for extra density of DNA at exit side of SNF2h-nucleosome complexes.

(A–C) Cryo-EM density map of the nucleosome either with SNF2h bound at (A) SHL-2 and (B) SHL+2 with 140 mM KCl with the simulated density map of nucleosome without two additional base-pairs filtered to a common resolution (A: 3.9 Å, B: 6.9 Å). (C) Cryo-EM density map of nucleosome without added SNF2h with the simulated density map of nucleosome without two additional base-pairs filtered to a common resolution (7.4 Å). For A–C), left: each panel shows an overview of the EM density (light blue) overlaid onto the simulated nucleosome map. Top right: enlarged view centered on the DNA exit site. Bottom right: enlarged view of the DNA exit site showing the difference density between the simulated map and the EM density at σ = 7. (D) Left: cryo-EM density map of doubly-bound nucleosome with 70 mM KCl is overlaid with the cryo-EM density map of the simulated nucleosome map. Both maps are filtered to a resolution of 8.4 Å. Top right: an enlarged view of the DNA end. Bottom right: density map of nucleosome alone is overlaid with the difference density calculated between the two, and visualized at σ = 7.

Figure 1—figure supplement 6. Bootstrapped maps of SNF2h-nucleosome complex.

20 bootstrapped maps were calculated by using 20 subsets of 5000 particles bootstrapped from the particles that were used to calculate the 3.9 Å reconstruction. Slices through the variance map at different sigma levels (2, 3, 4 and 5) are shown, and reveal no significant variance. Transparent overlay of the SHL-2 side 3.9 Å density is shown for orientation.