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- EMDB-8949: Cryo-EM structure of the centromeric nucleosome (Native alpha sat... -

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Basic information

Entry
Database: EMDB / ID: EMD-8949
TitleCryo-EM structure of the centromeric nucleosome (Native alpha satellite DNA) in complex with a single chain antibody fragment
Map datamain map
Sample
  • Complex: CENP-A nucleosome and antibody complex
    • Protein or peptide: Histone H3-like centromeric protein A
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
    • Protein or peptide: scFvSingle-chain variable fragment
Function / homology
Function and homology information


CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin ...CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / pericentric heterochromatin / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / telomere organization / RNA Polymerase I Promoter Opening / SUMOylation of chromatin organization proteins / Assembly of the ORC complex at the origin of replication / DNA methylation / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / innate immune response in mucosa / PRC2 methylates histones and DNA / Defective pyroptosis / RHO GTPases Activate Formins / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / G2/M DNA damage checkpoint / B-WICH complex positively regulates rRNA expression / HDMs demethylate histones / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / structural constituent of chromatin / Transcriptional regulation of granulopoiesis / Separation of Sister Chromatids / UCH proteinases / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / HATs acetylate histones / Processing of DNA double-strand break ends / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / killing of cells of another organism / Estrogen-dependent gene expression / defense response to Gram-negative bacterium / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / Amyloid fiber formation / protein heterodimerization activity / negative regulation of cell population proliferation / chromatin binding / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / nucleus / cytosol
Similarity search - Function
Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site ...Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H3-like centromeric protein A / Histone H4
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 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.
History
DepositionJul 6, 2018-
Header (metadata) releaseOct 10, 2018-
Map releaseMay 22, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing 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
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  • 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-6e0p
  • Surface level: 0.014
  • 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_8949.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Voxel sizeX=Y=Z: 1.005 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.07776524 - 0.16283152
Average (Standard dev.)0.00000024984 (±0.0043129544)
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-0.0780.1630.000

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Supplemental data

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Mask #1

Fileemd_8949_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #1

Fileemd_8949_additional.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map1

Fileemd_8949_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map2

Fileemd_8949_half_map_2.map
Annotationhalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : CENP-A nucleosome and antibody complex

EntireName: CENP-A nucleosome and antibody complex
Components
  • Complex: CENP-A nucleosome and antibody complex
    • Protein or peptide: Histone H3-like centromeric protein A
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
    • Protein or peptide: scFvSingle-chain variable fragment

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Supramolecule #1: CENP-A nucleosome and antibody complex

SupramoleculeName: CENP-A nucleosome and antibody complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Histone H3-like centromeric protein A

MacromoleculeName: Histone H3-like centromeric protein A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.038818 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSHHHHHH SSGLPRSHMG PRRRSRKPEA PRRRSPSPTP TPGPSRRGPS LGASSHQHSR RRQGWLKEIR KLQKSTHLLI RKLPFSRLA REICVKFTRG VDFNWQAQAL LALQEAAEAF LVHLFEDAYL LTLHAGRVTL FPKDVQLARR IRGLEEGLG

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.394426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVY ALKRQGRTLY GFGG

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Macromolecule #3: Histone H2A type 1-B/E

MacromoleculeName: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.165551 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK ARAKAKTRSS RAGLQFPVGR VHRLLRKGNY SERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIR NDEELNKLLG RVTIAQGGVL PNIQAVLLPK KTESHHKAKG K

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Macromolecule #4: Histone H2B type 1-J

MacromoleculeName: Histone H2B type 1-J / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.935239 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSI YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSAK

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Macromolecule #7: scFv

MacromoleculeName: scFv / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 29.030146 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKSSHHHHHH ENLYFQSNAM EVQLQQSGPE LVEPGTSVKM PCKASGYTFT SYTIQWVKQT PRQGLEWIGY IYPYNAGTKY NEKFKGKAT LTSDKSSSTV YMELSSLTSE DSAVYYCARK SSRLRSTLDY WGQGTSVTVS SGGGGSGGGG SGGGGSMDIK M TQSPSSMH ...String:
MKSSHHHHHH ENLYFQSNAM EVQLQQSGPE LVEPGTSVKM PCKASGYTFT SYTIQWVKQT PRQGLEWIGY IYPYNAGTKY NEKFKGKAT LTSDKSSSTV YMELSSLTSE DSAVYYCARK SSRLRSTLDY WGQGTSVTVS SGGGGSGGGG SGGGGSMDIK M TQSPSSMH ASLGERVTIT CKASQDIRSY LSWYQQKPWK SPKTLIYYAT SLADGVPSRF SGSGSGQDFS LTINNLESDD TA TYYCLQH GESPYTFGSG TKLEIKRA

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Macromolecule #5: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.539535 KDa
SequenceString: (DA)(DT)(DC)(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) ...String:
(DA)(DT)(DC)(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)(DG) (DA)(DT)

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Macromolecule #6: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.948793 KDa
SequenceString: (DA)(DT)(DC)(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) ...String:
(DA)(DT)(DC)(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)(DG) (DA)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: blot for 2.5 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-38 / Number grids imaged: 1 / Number real images: 1416 / Average exposure time: 15.2 sec. / Average electron dose: 40.0 e/Å2
Details: Images were collected in movie-mode at 38 frames over 15.2 seconds
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 8 / Avg.num./class: 38000 / Software - Name: RELION (ver. 3.0 beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta2)
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0 beta2) / Number images used: 301644
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6e0p:
Cryo-EM structure of the centromeric nucleosome (Native alpha satellite DNA) in complex with a single chain antibody fragment

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