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- EMDB-18775: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box -

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Basic information

Entry
Database: EMDB / ID: EMD-18775
TitleHuman CENP-A nucleosome assembled on 601 DNA with CENP-B box
Map dataHuman CENP-A nucleosome assembled on 601 DNA with CENP-B box
Sample
  • Complex: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box
    • Protein or peptide: Human histone CENP-A
    • Protein or peptide: Human histone H4
    • Protein or peptide: Human histone H2A
    • Protein or peptide: Human histone H2B
    • Protein or peptide: Human CENP-B
    • DNA: 601 synthetic DNA
KeywordsNucleosome / centromeric / CENP-A / Histones / Human / DNA / DNA BINDING PROTEIN / 601 / synthetic DNA
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsAli-Ahmad A / Sekulic N
Funding support Norway, 2 items
OrganizationGrant numberCountry
Research Council of Norway187615 Norway
Research Council of Norway325528 Norway
CitationJournal: Nat Commun / Year: 2023
Title: CENP-A and CENP-B collaborate to create an open centromeric chromatin state.
Authors: Harsh Nagpal / Ahmad Ali-Ahmad / Yasuhiro Hirano / Wei Cai / Mario Halic / Tatsuo Fukagawa / Nikolina Sekulić / Beat Fierz /
Abstract: Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore ...Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.
History
DepositionOct 27, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18775.png

Downloads & links

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Map

FileDownload / File: emd_18775.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman CENP-A nucleosome assembled on 601 DNA with CENP-B box
Voxel sizeX=Y=Z: 1.8675 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.5088532 - 1.0756431
Average (Standard dev.)0.0009456074 (±0.037897035)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18775_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18775_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18775_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human CENP-A nucleosome assembled on 601 DNA with CENP-B box

EntireName: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box
Components
  • Complex: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box
    • Protein or peptide: Human histone CENP-A
    • Protein or peptide: Human histone H4
    • Protein or peptide: Human histone H2A
    • Protein or peptide: Human histone H2B
    • Protein or peptide: Human CENP-B
    • DNA: 601 synthetic DNA

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Supramolecule #1: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box

SupramoleculeName: Human CENP-A nucleosome assembled on 601 DNA with CENP-B box
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Human histone CENP-A

MacromoleculeName: Human histone CENP-A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRKLQKSTH LLIRKLPFSR LAREICVKFT RGVDFNWQAQ ALLALQEAAE AFLVHLFEDA YLLTLHAGRV TLFPKDVQLA RRIRGLEEGL G

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Macromolecule #2: Human histone H4

MacromoleculeName: Human histone H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #3: Human histone H2A

MacromoleculeName: Human histone H2A / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GPLGMSGRGK QGGKARAKAK SRSSRAGLQF PVGRVHRLLR KGNYAERVGA GAPVYMAAVL EYLTAEILEL AGNAARDNKK TRIIPRHLQL AIRNDEELNK LLGKVTIAQG GVLPNIQAVL LPKKTESHHK AKGK

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Macromolecule #4: Human histone H2B

MacromoleculeName: Human histone H2B / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSV YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRSTI TSREIQTAVR LLLPGELAKH AVSEGTKAVT KYTSSK

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Macromolecule #5: Human CENP-B

MacromoleculeName: Human CENP-B / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DSLEFIASKL AGPKRRQLTF REKSRIIQEV EENPDLRKGE IARRFNIPPS TLSTILKNKR AILASERKYG VASTCRKTNK LSPYDKLEGL LIAWFQQIRA AGLPVKGIIL KEKALRIAEE LGMDDFTASN GWLDRFRRRH GVVSCSGVAR ARARNAAPRT PAAPASPAAV ...String:
DSLEFIASKL AGPKRRQLTF REKSRIIQEV EENPDLRKGE IARRFNIPPS TLSTILKNKR AILASERKYG VASTCRKTNK LSPYDKLEGL LIAWFQQIRA AGLPVKGIIL KEKALRIAEE LGMDDFTASN GWLDRFRRRH GVVSCSGVAR ARARNAAPRT PAAPASPAAV PSEGSGGSTT GWRAREEQPP SVAEGYASQD VFSATETSLW YDFLPDQAAG LCGGDGRPRQ ATQRLSVLLC ANADGSEKLP PLVAGKSAKP RAGQAGLPCD YTANSKGGVT TQALAKYLKA LDTRMAAESR RVLLLAGRLA AQSLDTSGLR HVQLAFFPPG TVHPLERGVV QQVKGHYRQA MLLKAMAALE GQDPSGLQLG LTEALHFVAA AWQAVEPSDI AACFREAGFG GGPNATITTS LKSEGEEEEE EEEEEEEEEG EGEEEEEEGE EEEEEGGEGE ELGEEEEVEE EGDVDSDEEE EEDEESSSEG LEAEDWAQGV VEAGGSFGAY GAQEEAQCPT LHFLEGGEDS DSDSEEEDDE EEDDEDEDDD DDEEDGDEVP VPSFGEAMAY FAMVKRYLTS FPIDDRVQSH ILHLEHDLVH VTRKNHARQA GVRGLGHQSP HHHHHH

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Macromolecule #6: 601 synthetic DNA

MacromoleculeName: 601 synthetic DNA / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
GATCGGTCTC ATAGCCTTCG TTGGAAACGG GAGAATCCCG GTGCCGAGGC CGCTCAATTG GTCGTAGACA GCTCTAGCAC CGCTTAAACG CACGTACGCG CTGTCCCCCG CGTTTTAACC GCCAAGGGGA TTACTCCCTA GTCTCCAGGC ACGTGTCAGA TATATACATC CTGTCACACT GTGGATC

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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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 / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 246772
FSC plot (resolution estimation)

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