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- EMDB-8945: Cryo-EM structure of the CENP-A nucleosome (W601) in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-8945
TitleCryo-EM structure of the CENP-A nucleosome (W601) in complex with a single chain antibody fragment
Map data
SampleCENP-A nucleosome (W601) complex:
Histone H3-like centromeric protein A / Histone H4 / Histone H2A type 1-B/E / Histone H2B type 1-J / (nucleic-acidNucleic acid) x 2 / scFvSingle-chain variable fragment
Function / homology
Function and homology information


condensed chromosome inner kinetochore / protein localization to chromosome, centromeric region / kinetochore assembly / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / negative regulation of megakaryocyte differentiation / condensed nuclear chromosome, centromeric region / CENP-A containing nucleosome assembly / establishment of mitotic spindle orientation / DNA replication-independent nucleosome assembly ...condensed chromosome inner kinetochore / protein localization to chromosome, centromeric region / kinetochore assembly / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / negative regulation of megakaryocyte differentiation / condensed nuclear chromosome, centromeric region / CENP-A containing nucleosome assembly / establishment of mitotic spindle orientation / DNA replication-independent nucleosome assembly / chromosome, centromeric region / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / mitotic cytokinesis / innate immune response in mucosa / telomere organization / DNA replication-dependent nucleosome assembly / nuclear nucleosome / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / nuclear chromosome / DNA-templated transcription, initiation / regulation of megakaryocyte differentiation / protein heterotetramerization / nucleosome assembly / nucleosome / lipopolysaccharide binding / double-strand break repair via nonhomologous end joining / chromatin organization / nuclear chromosome, telomeric region / killing of cells of other organism / antibacterial humoral response / antimicrobial humoral immune response mediated by antimicrobial peptide / protein ubiquitination / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / nuclear chromatin / protein domain specific binding / chromatin binding / cellular protein metabolic process / viral process / protein heterodimerization activity / protein-containing complex / RNA binding / cell / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / nucleus / cytosol
Histone H4 / Histone H4, conserved site / Histone H3/CENP-A / Histone H2B / Histone H2A / TATA box binding protein associated factor (TAF) / CENP-T/Histone H4, histone fold / Histone H2A/H2B/H3 / Histone-fold / Histone H2A conserved site / Histone H2A, C-terminal domain
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H3-like centromeric protein A / Histone H4
Biological speciesHomo sapiens (human) / Escherichia coli (E. coli) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsYadav KNS / Zhou B-R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)the intramural research program United States
CitationJournal: Nat Commun / Year: 2019
Title: Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment.
Authors: Bing-Rui Zhou / K N Sathish Yadav / Mario Borgnia / Jingjun Hong / Baohua Cao / Ada L Olins / Donald E Olins / Yawen Bai / Ping Zhang /
Abstract: Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a ...Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.
Validation ReportPDB-ID: 6e0c

SummaryFull reportAbout validation report
History
DepositionJul 6, 2018-
Header (metadata) releaseOct 31, 2018-
Map releaseMay 22, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6e0c
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8945.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 257.28 Å
1.01 Å/pix.
x 256 pix.
= 257.28 Å
1.01 Å/pix.
x 256 pix.
= 257.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.005 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.05282611 - 0.1300725
Average (Standard dev.)0.0000007817 (±0.0036126522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 257.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0051.0051.005
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z257.280257.280257.280
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0530.1300.000

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Supplemental data

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Segmentation: #1

Fileemd_8945_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire CENP-A nucleosome (W601) complex

EntireName: CENP-A nucleosome (W601) complex / Number of components: 8

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Component #1: protein, CENP-A nucleosome (W601) complex

ProteinName: CENP-A nucleosome (W601) complex / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Histone H3-like centromeric protein A

ProteinName: Histone H3-like centromeric protein A / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 18.038818 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.394426 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Histone H2A type 1-B/E

ProteinName: Histone H2A type 1-B/E / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.165551 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Histone H2B type 1-J

ProteinName: Histone H2B type 1-J / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.935239 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: nucleic-acid, DNA (146-MER)

nucleic acidName: DNA (146-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC) (DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG)(DT)(DA)(DA)(DT)(DC)(DC)(DC)(DC)(DT) (DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT)(DA)(DA) (DA)(DA)(DC)(DG)(DC)(DG)(DG)(DG)(DG)(DG) (DA)(DC)(DA)(DG)(DC)(DG)(DC)(DG)(DT)(DA) (DC)(DG)(DT)(DG)(DC)(DG)(DT)(DT)(DT)(DA) (DA)(DG)(DC)(DG)(DG)(DT)(DG)(DC)(DT)(DA) (DG)(DA)(DG)(DC)(DT)(DG)(DT)(DC)(DT)(DA) (DC)(DG)(DA)(DC)(DC)(DA)(DA)(DT)(DT)(DG) (DA)(DG)(DC)(DG)(DG)(DC)(DC)(DT)(DC)(DG) (DG)(DC)(DA)(DC)(DC)(DG)(DG)(DG)(DA)(DT) (DT)(DC)(DT)(DC)(DG)(DA)(DT)
MassTheoretical: 45.610043 kDa
SourceSpecies: Escherichia coli (E. coli)

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Component #7: nucleic-acid, DNA (146-MER)

nucleic acidName: DNA (146-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DG)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA)(DG) (DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA)(DA) (DA)(DC)(DG)(DC)(DA)(DC)(DG)(DT)(DA)(DC) (DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC)(DC)(DC) (DC)(DC)(DG)(DC)(DG)(DT)(DT)(DT)(DT)(DA) (DA)(DC)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DT)(DT)(DA)(DC)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC)(DC)(DA) (DG)(DG)(DC)(DA)(DC)(DG)(DT)(DG)(DT)(DC) (DA)(DG)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DC) (DA)(DT)(DC)(DC)(DG)(DA)(DT)
MassTheoretical: 45.13877 kDa
SourceSpecies: Escherichia coli (E. coli)

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Component #8: protein, scFv

ProteinName: scFvSingle-chain variable fragment / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 29.030146 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 494510
3D reconstructionSoftware: RELION / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3AN2
Output model

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