|Entry||Database: EMDB / ID: EMD-0772|
|Title||H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA|
|Sample||H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 15.7 Å|
|Authors||Takizawa Y / Ho C-H / Tachiwana H / Ohi M / Wolf M / Kurumizaka H|
|Funding support|| Japan, 6 items |
|Citation||Journal: Structure / Year: 2019|
Title: Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome.
Authors: Yoshimasa Takizawa / Cheng-Han Ho / Hiroaki Tachiwana / Hideyuki Matsunami / Wataru Kobayashi / Midori Suzuki / Yasuhiro Arimura / Tetsuya Hori / Tatsuo Fukagawa / Melanie D Ohi / Matthias Wolf / Hitoshi Kurumizaka /
Abstract: The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric ...The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_0772.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.4 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA
|Entire||Name: H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA|
Number of components: 1
-Component #1: protein, H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA
|Protein||Name: H3-H3-H3 tri-nucleosome with the 30 base-pair linker DNA|
Recombinant expression: No
|Mass||Theoretical: 600 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.03 mg/mL / pH: 7.8|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Imaging||Microscope: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON III (4k x 4k)|
|Image acquisition||Number of digital images: 8035|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 4534|
|3D reconstruction||Software: RELION / Resolution: 15.7 Å / Resolution method: FSC 0.143 CUT-OFF|
|FSC plot (resolution estimation)|
-Atomic model buiding
|Modeling #1||Refinement protocol: rigid body|
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