the cryo-EM density of the 30-nm chromatin fiber
[EM Data Bank (EMDB) 2601] (fig. S11, A and B)
(9). We simulated an EM density for the atomic
structure of chicken linker histone H5 bound to
linker DNAs at the dyad of a DNA–core nucleosome particle [Protein Data Bank (PDB) 4QLC]
(67) (fig. S11A) and then used Sculptor to simultaneously fit 12 nucleosome–linker histone units
into the 30-nm fiber density map. The Sculptor
model closely matches the experimentally determined known nucleosome organization of the
30-nm chromatin fiber (fig. S11B, middle panel),
validating our approach.
We then applied a similar approach to model
nucleosome–linker histone H5 units in chromatin-
chain EMT densities (Fig. 5, D, E, and H). The
EMT volumes of chromatin chains were used as
a starting point to estimate the initial number of
nucleosome–linker histone units to model. Sculp-
tor alters the position of each nucleosome–linker
histone H5 unit for 500 generations (64). We ran
five independent Sculptor evolutions for each
chromatin-chain EMT density (fig. S11, C to E).
All of the Sculptor nucleosome–linker histone
models have cross-correlation values between
80 and 92% (Fig. 5, J to L, and fig. S11, C to E).
Not surprisingly, there are differences between
each of the solutions as well as unaccounted for
densities. The unmatched densities may reflect
off-dyad H1 binding (67), histone-tail structures,
or proteins other than nucleosomes that interact
with DNA and chromatin in the nucleus, such as
high-mobility groups (HMGs) (68). In addition,
Ou et al., Science 357, eaag0025 (2017) 28 July 2017 5 of 13
Fig. 2. ChromEM: DRAQ5 excitation photo-oxidizes DAB on DNA in the
nucleus and enables chromatin to be visualized by osmium staining in
EM. (A) U2OS cells were fixed with glutaraldehyde, stained with DRAQ5,
and DAB photo-oxidized for 7 min. Cells were then stained with OsO4
and thin sections prepared for TEM. EtOH, ethanol. Scale bar, 10 mm.
(B to E) Magnified views of the cells in the labeled rectangles in panel
(A). TEM images of a cell outside the photo-oxidation field (B), inside
the photo-oxidation field (C), and bisected by the excitation field
(D) and (E). Zoom-in (red box) is shown to the right. Nuclear membrane
(arrow) and nucleolus (double arrow) are shown. Scale bar, 2 mm.