DOI: 10.5061/dryad.ttdz08m21
title: Data from: Cryo-electron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination

description: Nucleosome chains fold and self-associate to form higher order structures
 whose internal organization is unknown. Here, cryo-electron tomography
 (cryo-ET) of native human chromatin reveals novel folding motifs such as
 1) non-uniform nucleosome stacking, 2) intermittent parallel and
 perpendicular orientations of adjacent nucleosome planes, and 3) an
 inverse zigzag nucleosome chain path, which deviates from the direct
 zigzag topology seen in reconstituted nucleosomal arrays. By examining
 these self-associated structures, we observed prominent nucleosome
 stacking in-cis and anti-parallel nucleosome interactions in-trans, which
 are consistent with partial nucleosome interdigitation. Histone
 citrullination strongly inhibits nucleosome stacking and self-association
 with a modest effect on chromatin folding, while the reconstituted arrays
 showed a zigzag topology which undergoes a dramatic unfolding induced by
 histone citrullination. This study sheds light on the internal structure
 of compact chromatin nanoparticles and suggests a novel mechanism for how
 epigenetic changes in chromatin are retained across both open and
 condensed forms of chromatin.
description: Cryo-Electron microscopy and tomographic reconstruction
 Chromatin samples incubated for 20 min. without or with
 appropriate concentrations of MgCl2 were mixed with a suspension of 10 nm
 fiduciary gold particles (Sigma Aldrich # 741957), which were coated in
 bovine serum albumin to prevent clustering. 3 ul chromatin samples with a
 concentration of about 0.2 mg/ml DNA were applied to Quantifoil R2/2 200
 mesh copper grids (EMS Q250-CR2). Vitrification was conducted by plunging
 into liquid ethane using our FEI Vitrobot Mk IV Grid Plunging System at
 100% humidity, 4o C, and setting the blotting strength at 5, and blotting
 time at 3.5 sec. Imaging of the vitrified samples was conducted on Titan
 Krios G3i 300 kV electron microscope, equipped with a K3 direct electron
 detector (Gatan, CA) at the Penn State Hershey cryo-EM core. We used
 Tomography-5.7.1. software (Thermo Fisher) for controlling data
 acquisition and collecting tilt-series. CryoEM tilt series (± 60o ) were
 collected at 5o intervals in dose symmetric mode, at either 2.2
 angstroms/pixel (defocus -6 um) or 1.7 angstroms/pixel (defocus -5 um),
 with zero-loss peak energy filtration through a 20-eV slit. Images were
 collected in 1x counting mode. Each tilt series had a cumulative dose of
 120 electrons/Å2. Tilt series were aligned using fiducials in the IMOD
 software suite (https://bio3d.colorado.edu/imod/), CTF corrected and SIRT
 reconstructed. Centroid/axis/plane (CAP) modeling of
 nucleosome chain folding The reconstructed un-binned
 tomograms were visualized and segmented into smaller subtomograms by
 IMOD/3dmod. Each volume was inverted using “newstack” to generate
 subtomograms with positive intensity corresponding to high density and
 filtered using IMOD command: “nad_eed_3d -n 30 -f -k 50” to reduce noise
 and enhance chromatin edges. The filtered subtomograms were exported into
 UCSF CHIMERA (RBVI, Univ. San Francisco, CA) for interactive visualization
 and analysis of nucleosome structures. In CHIMERA, the filtered volumes
 were fitted with nucleosome core X-ray crystal structure (pdb 2CV5)
 semi-automatically to correspondent electron densities in the volume using
 the ‘fitmap’ command. The fitting of each nucleosome followed the local
 maximal electron density and thus was independent from the observer’s
 bias. After fitting, the nucleosomes were overlaid with centroids
 selecting the centers of masses, center-to-center axes, and nucleosome
 planes crossing the nucleosome at the dyad axis using the structure
 analysis ‘Axels/Planes/Centroids’ tool. For stereological analysis, each
 nucleosome in an array was numbered, if possible, starting from one end of
 the array and numbering consecutive nucleosomes to the other end of the
 array. Where linkers were not visible (such as in the tertiary chromatin
 structures), the nucleosomes were labeled based on their spatial
 proximity. The following measurements were recorded from such
 Centroid-Axial-Plane (CAP) models for each nucleosome (n) in an array: a)
 center-to-center distance D to the next nucleosome (n+1) in the array, b)
 center-to-center distance N to the nearest nucleosome (nx) in the 3D
 space, angle a between the two axes connecting each nucleosome with the
 previous one (n-1) and the next one (n+1) in a chain, angle b between the
 planes of consecutive nucleosomes n and n+1, and an angle para between the
 plane of each nucleosome (n) and the plane of the nearest nucleosome (nx)
 in the 3D space (see Fig 3 B). All distance and angle measurements for
 individual nucleosome arrays are included in the Suppl. Table 2. The
 absolute nucleosome proximity values were obtained by subtracting the
 number of each nucleosome (n) from the number of its nearest nucleosome
 (nx) in the 3D space. The measured angle and plane values were analyzed
 statistically to determine the distribution profiles and average values
 that discriminate between the condensed and open nucleosomes arrays.
 Distance N and angle para were recorded for all nucleosomes. For some
 nucleosomes condensed by 0.75 mM Mg2+ (~21% of all nucleosomes) and for
 all nucleosomes condensed at &gt; 1 mM Mg2+, the linkers were not
 resolved and corresponding distances D and angles alpha and beta were
 excluded from the statistical analysis. SDs were obtained from at least
 three tomograms and at least two independently cultured biological
 samples; p- values represent probability associated with a Student’s
 two-sample unequal variance t-test with a two-tailed
 distribution.
description: This data is separated into two different folders.
 1) tilt_series_data - Contains all the original cryo-EM tilt
 series data used for analysis in the paper. To view the
 data, you will need to download and install IMOD: https://bio3d.colorado.edu/imod/ To view a single tilt series use the “3dmod” . Like this, 3dmod tilt_series_name.mrc 2) CAP_models - Each subfolder contains folders with both the filtered .MRC volume and its corresponding .PY file. To open this data you will need to download and install Chimera. https://www.cgl.ucsf.edu/chimera/ To view the data and the model, open Chimera and use it to open the .PY file within one of the directories. It should load the corresponding .MRC and display the placement of each centroid, axis and plane within it.
description: This data is separated into two different folders. 1. tilt_series_data -
 Contains all the original cryo-EM tilt series data used for analysis in
 the paper. To view the data, you will need to download and install IMOD:
 To view a single tilt series use
 the “3dmod” . Like this, 3dmod tilt_series_name.mrc 1. CAP_models - Each
 subfolder contains folders with both the filtered .MRC volume and its
 corresponding .PY file. To open this data you will need to download and
 install Chimera. To view the
 data and the model, open Chimera and use it to open the .PY file within
 one of the directories. It should load the corresponding .MRC and display
 the placement of each centroid, axis and plane within it.