National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
R01HL115153
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM104427
United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)
R01MH077303
United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)
R01DK042667
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM127018
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM032543
United States
Citation
Journal: Mol Cell / Year: 2022 Title: Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography. Authors: Meng Zhang / César Díaz-Celis / Bibiana Onoa / Cristhian Cañari-Chumpitaz / Katherinne I Requejo / Jianfang Liu / Michael Vien / Eva Nogales / Gang Ren / Carlos Bustamante / Abstract: It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, ...It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.
Download / File: emd_24905.map.gz / Format: CCP4 / Size: 200 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Grown spherical tetranucleosome condensate (2)
Voxel size
X=Y=Z: 11.68 Å
Density
Minimum - Maximum
-0.83800703 - 3.634139
Average (Standard dev.)
0.0054754666 (±0.06163078)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
512
512
200
Spacing
512
512
200
Cell
A: 5980.16 Å / B: 5980.16 Å / C: 2336.0 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
-
Entire : Larger spherical type of tetranucleosome condensates
Entire
Name: Larger spherical type of tetranucleosome condensates
Components
Complex: Larger spherical type of tetranucleosome condensates
-
Supramolecule #1: Larger spherical type of tetranucleosome condensates
Supramolecule
Name: Larger spherical type of tetranucleosome condensates / type: complex / ID: 1 / Parent: 0 Details: Spherical condensates formed at physiological salt condition (20 mM HEPES-KOH pH 7.5; 150 mM NaCl; 5 mM MgCl2; 1 mM DTT) after 10 min incubation.
Source (natural)
Organism: Xenopus laevis (African clawed frog)
Recombinant expression
Organism: Escherichia coli (E. coli)
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Experimental details
-
Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
particle
-
Sample preparation
Buffer
pH: 7.5
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 %
Sectioning
Other: NO SECTIONING
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 40 / Average exposure time: 2.5 sec. / Average electron dose: 7.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
-
Image processing
Final reconstruction
Number images used: 40
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