Cryo-electron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination

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 ..., 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 interval..., 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., 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.