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- EMDB-9251: CENP-A nucleosome bound by two copies of CENP-C(CD) and two copie... -

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Basic information

Entry
Database: EMDB / ID: EMD-9251
TitleCENP-A nucleosome bound by two copies of CENP-C(CD) and two copies CENP-N(NT)
Map data
SampleCENP-A chromatin complex bound with CENP-C and CENP-N of CCAN kinetochore components:
Histone H3-like centromeric protein A / Histone H4 / Histone H2A type 1-C / Histone H2B type 2-F / (nucleic-acidNucleic acid) x 2 / (Centromere protein ...) x 2
Function / homology
Function and homology information


monopolar spindle attachment to meiosis I kinetochore / condensed chromosome inner kinetochore / centromeric DNA binding / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / protein localization to chromosome, centromeric region / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / condensed nuclear chromosome, centromeric region / negative regulation of megakaryocyte differentiation ...monopolar spindle attachment to meiosis I kinetochore / condensed chromosome inner kinetochore / centromeric DNA binding / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / protein localization to chromosome, centromeric region / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / condensed nuclear chromosome, centromeric region / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / establishment of mitotic spindle orientation / condensed chromosome kinetochore / DNA replication-independent nucleosome assembly / telomere capping / pericentric heterochromatin / chromosome, centromeric region / chromatin silencing / mitotic cytokinesis / telomere organization / DNA replication-dependent nucleosome assembly / nuclear nucleosome / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / kinetochore / regulation of gene silencing by miRNA / nuclear chromosome / DNA-templated transcription, initiation / chromosome segregation / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / midbody / mitotic cell cycle / double-strand break repair via nonhomologous end joining / chromatin organization / nuclear chromosome, telomeric region / nuclear body / protein heterodimerization activity / cell division / negative regulation of cell population proliferation / nuclear chromatin / protein domain specific binding / chromatin binding / cellular protein metabolic process / viral process / host cell nucleus / protein-containing complex / go:0005623: / RNA binding / DNA binding / extracellular exosome / membrane / extracellular region / nucleoplasm / identical protein binding / nucleus / cytosol
Histone-fold / Histone H4 / Histone H2B / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Centromere protein Chl4/mis15/CENP-N / RmlC-like cupin domain superfamily / RmlC-like jelly roll fold / Histone H4, conserved site / Mif2/CENP-C cupin domain ...Histone-fold / Histone H4 / Histone H2B / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Centromere protein Chl4/mis15/CENP-N / RmlC-like cupin domain superfamily / RmlC-like jelly roll fold / Histone H4, conserved site / Mif2/CENP-C cupin domain / Kinetochore assembly subunit CENP-C, N-terminal domain / Centromere protein C/Mif2/cnp3 / CENP-C, middle DNMT3B-binding domain / Histone H3/CENP-A / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Histone H2A
Histone H3-like centromeric protein A / Histone H4 / Centromere protein C / Histone H2B type 2-F / Histone H2A type 1-C / Centromere protein N
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAllu PK / Black BE
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM123233 GM130302 United States
CitationJournal: Curr. Biol. / Year: 2019
Title: Structure of the Human Core Centromeric Nucleosome Complex.
Authors: Praveen Kumar Allu / Jennine M Dawicki-McKenna / Trevor Van Eeuwen / Moriya Slavin / Merav Braitbard / Chen Xu / Nir Kalisman / Kenji Murakami / Ben E Black /
Abstract: Centromeric nucleosomes are at the interface of the chromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the ...Centromeric nucleosomes are at the interface of the chromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the histone H3 variant, CENP-A, and its binding proteins, CENP-C (through its central domain; CD) and CENP-N (through its N-terminal domain; NT). CENP-C can engage nucleosomes through two domains: the CD and the CENP-C motif (CM). CENP-C is part of the CCNC by virtue of its high specificity for CENP-A nucleosomes and ability to stabilize CENP-A at the centromere. CENP-C is thought to engage a neighboring nucleosome, either one containing conventional H3 or CENP-A, and a crystal structure of a nucleosome complex containing two copies of CENP-C was reported. Recent structures containing a single copy of CENP-N bound to the CENP-A nucleosome in the absence of CENP-C were reported. Here, we find that one copy of CENP-N is lost for every two copies of CENP-C on centromeric chromatin just prior to kinetochore formation. We present the structures of symmetric and asymmetric forms of the CCNC that vary in CENP-N stoichiometry. Our structures explain how the central domain of CENP-C achieves its high specificity for CENP-A nucleosomes and how CENP-C and CENP-N sandwich the histone H4 tail. The natural centromeric DNA path in our structures corresponds to symmetric surfaces for CCNC assembly, deviating from what is observed in prior structures using artificial sequences. At mitosis, we propose that CCNC asymmetry accommodates its asymmetric connections at the chromosome/kinetochore interface. VIDEO ABSTRACT.
Validation ReportPDB-ID: 6mup

SummaryFull reportAbout validation report
History
DepositionOct 23, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseJul 24, 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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mup
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9251.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.14778884 - 0.25917518
Average (Standard dev.)0.00084960175 (±0.008206707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1480.2590.001

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Supplemental data

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Additional map: CENP-A nucleosome bound by two copies of CENP-C(CD)...

Fileemd_9251_additional.map
AnnotationCENP-A nucleosome bound by two copies of CENP-C(CD) and two copies CENP-N(NT)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire CENP-A chromatin complex bound with CENP-C and CENP-N of CCAN kin...

EntireName: CENP-A chromatin complex bound with CENP-C and CENP-N of CCAN kinetochore components
Number of components: 9

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Component #1: cellular-component, CENP-A chromatin complex bound with CENP-C an...

Cellular-componentName: CENP-A chromatin complex bound with CENP-C and CENP-N of CCAN kinetochore components
Recombinant expression: No
MassExperimental: 276 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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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: 11.993037 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #3: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 10.604521 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #4: protein, Histone H2A type 1-C

ProteinName: Histone H2A type 1-C / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.494393 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #5: protein, Histone H2B type 2-F

ProteinName: Histone H2B type 2-F / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 10.249723 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #6: nucleic-acid, DNA (147-MER)

nucleic acidName: DNA (147-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DA)(DT)(DC)(DA)(DA)(DA)(DT)(DA)(DT)(DC) (DC)(DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA) (DT)(DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA) (DA)(DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG) (DG)(DA)(DA)(DA)(DC)(DT) ...Sequence:
(DA)(DT)(DC)(DA)(DA)(DA)(DT)(DA)(DT)(DC) (DC)(DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA) (DT)(DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA) (DA)(DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG) (DG)(DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC) (DC)(DA)(DT)(DC)(DA)(DA)(DA)(DA)(DG)(DG) (DC)(DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC) (DT)(DC)(DT)(DG)(DT)(DG)(DA)(DG)(DT)(DG) (DA)(DA)(DA)(DC)(DT)(DC)(DC)(DA)(DT)(DC) (DA)(DT)(DC)(DA)(DC)(DA)(DA)(DA)(DG)(DA) (DA)(DT)(DA)(DT)(DT)(DC)(DT)(DG)(DA)(DG) (DA)(DA)(DT)(DG)(DC)(DT)(DT)(DC)(DC)(DG) (DT)(DT)(DT)(DG)(DC)(DC)(DT)(DT)(DT)(DT) (DA)(DT)(DA)(DT)(DG)(DA)(DA)(DC)(DT)(DT) (DC)(DC)(DT)(DC)(DG)(DA)(DT)
MassTheoretical: 45.141918 kDa
SourceSpecies: Homo sapiens (human)

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Component #7: nucleic-acid, DNA (147-MER)

nucleic acidName: DNA (147-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DA)(DT)(DC)(DG)(DA)(DG)(DG)(DA)(DA)(DG) (DT)(DT)(DC)(DA)(DT)(DA)(DT)(DA)(DA)(DA) (DA)(DG)(DG)(DC)(DA)(DA)(DA)(DC)(DG)(DG) (DA)(DA)(DG)(DC)(DA)(DT)(DT)(DC)(DT)(DC) (DA)(DG)(DA)(DA)(DT)(DA) ...Sequence:
(DA)(DT)(DC)(DG)(DA)(DG)(DG)(DA)(DA)(DG) (DT)(DT)(DC)(DA)(DT)(DA)(DT)(DA)(DA)(DA) (DA)(DG)(DG)(DC)(DA)(DA)(DA)(DC)(DG)(DG) (DA)(DA)(DG)(DC)(DA)(DT)(DT)(DC)(DT)(DC) (DA)(DG)(DA)(DA)(DT)(DA)(DT)(DT)(DC)(DT) (DT)(DT)(DG)(DT)(DG)(DA)(DT)(DG)(DA)(DT) (DG)(DG)(DA)(DG)(DT)(DT)(DT)(DC)(DA)(DC) (DT)(DC)(DA)(DC)(DA)(DG)(DA)(DG)(DC)(DT) (DG)(DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT) (DT)(DT)(DT)(DG)(DA)(DT)(DG)(DG)(DA)(DG) (DC)(DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA) (DA)(DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT) (DG)(DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT) (DG)(DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT) (DA)(DT)(DT)(DT)(DG)(DA)(DT)
MassTheoretical: 45.582188 kDa
SourceSpecies: Homo sapiens (human)

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Component #8: protein, Centromere protein C

ProteinName: Centromere protein C / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.508834 kDa
SourceSpecies: Homo sapiens (human)

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Component #9: protein, Centromere protein N

ProteinName: Centromere protein N / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 25.052885 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL
Buffer solution: 10 mM HEPES, 50 mN NaCl, 1 mM EDTA, 1mM DTT
pH: 7.5
Support filmunspecified
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE / Details: Blot for 8 seconds before plunging.

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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: OTHER
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: OTHER / Defocus: 500.0 - 3000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 188995
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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