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- EMDB-48619: Structure of a native Drosophila melanogaster octameric nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-48619
TitleStructure of a native Drosophila melanogaster octameric nucleosome
Map dataRefined, unsharpened Coulomb potential density map. Particles extracted in 440x440 Fourier cropped to 360x360. Map refined in cryoSPARC
Sample
  • Complex: Natively purified octameric nucleosome from Drosophila melanogaster
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: Histone H3
    • Protein or peptide: Histone H4
    • DNA: DNA (164-MER)
    • DNA: DNA (164-MER)
KeywordsNucleosome / histones / histone / chromatin / DNA / GENE REGULATION
Function / homology
Function and homology information


HDMs demethylate histones / PKMTs methylate histone lysines / Interleukin-7 signaling / Chromatin modifying enzymes / Condensation of Prophase Chromosomes / SUMOylation of chromatin organization proteins / Metalloprotease DUBs / E3 ubiquitin ligases ubiquitinate target proteins / Factors involved in megakaryocyte development and platelet production / RCAF complex ...HDMs demethylate histones / PKMTs methylate histone lysines / Interleukin-7 signaling / Chromatin modifying enzymes / Condensation of Prophase Chromosomes / SUMOylation of chromatin organization proteins / Metalloprotease DUBs / E3 ubiquitin ligases ubiquitinate target proteins / Factors involved in megakaryocyte development and platelet production / RCAF complex / RMTs methylate histone arginines / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / polytene chromosome band / SIRT1 negatively regulates rRNA expression / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RNA Polymerase I Promoter Escape / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / HDACs deacetylate histones / Ub-specific processing proteases / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Regulation of endogenous retroelements by KRAB-ZFP proteins / larval somatic muscle development / Transcriptional regulation by small RNAs / Estrogen-dependent gene expression / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Senescence-Associated Secretory Phenotype (SASP) / HATs acetylate histones / UCH proteinases / Assembly of the ORC complex at the origin of replication / Oxidative Stress Induced Senescence / polytene chromosome / nucleosomal DNA binding / nuclear chromosome / heterochromatin formation / nucleosome assembly / structural constituent of chromatin / nucleosome / chromatin organization / chromosome / nucleic acid binding / protein heterodimerization activity / protein-containing complex binding / chromatin / DNA binding / nucleus
Similarity search - Function
: / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A ...: / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / 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 H2B / Histone H3 / Histone H4 / Histone H2A
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsVenette-Smith NL / Vishwakarma RK / Dollinger R / Schultz J / Venkatakrishnan V / Babitzke P / Anand G / Gilmour DS / Armache J-P / Murakami K
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM047477 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098399 United States
CitationJournal: To Be Published
Title: Structural Characterization of Native RNA Polymerase II Transcription Complexes in Drosophila melanogaster
Authors: Venette-Smith NL / Vishwakarma RK / Dollinger R / Schultz J / Venkatakrishnan V / Babitzke P / Anand G / Gilmour DS / Armache J-P / Murakami K
History
DepositionJan 13, 2025-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48619.png

Downloads & links

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Map

FileDownload / File: emd_48619.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined, unsharpened Coulomb potential density map. Particles extracted in 440x440 Fourier cropped to 360x360. Map refined in cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 360 pix.
= 415.368 Å		1.15 Å/pix.
x 360 pix.
= 415.368 Å		1.15 Å/pix.
x 360 pix.
= 415.368 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1538 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-0.0 - 2.4776704
Average (Standard dev.)0.005810098 (±0.038797896)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 415.368 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Refined, auto-sharpened Coulomb potential density map with Bfactor...

Fileemd_48619_additional_1.map
AnnotationRefined, auto-sharpened Coulomb potential density map with Bfactor = -50
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from cryoSPARC

Fileemd_48619_half_map_1.map
AnnotationHalf map 1 from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from cryoSPARC

Fileemd_48619_half_map_2.map
AnnotationHalf map 2 from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Natively purified octameric nucleosome from Drosophila melanogaster

EntireName: Natively purified octameric nucleosome from Drosophila melanogaster
Components
  • Complex: Natively purified octameric nucleosome from Drosophila melanogaster
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: Histone H3
    • Protein or peptide: Histone H4
    • DNA: DNA (164-MER)
    • DNA: DNA (164-MER)

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Supramolecule #1: Natively purified octameric nucleosome from Drosophila melanogaster

SupramoleculeName: Natively purified octameric nucleosome from Drosophila melanogaster
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: This entry represents a native octameric nucleosome (i.e. containing two H2A/H2B dimers and two H3/H4 dimers), purified from Drosophila melanogaster embryos
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 240 kDa/nm

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Macromolecule #1: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 11.552494 KDa
SequenceString:
AKSRSNRAGL QFPVGRIHRL LRKGNYAERV GAGAPVYLAA VMEYLAAEVL ELAGNAARDN KKTRIIPRHL QLAIRNDEEL NKLLSGVTI AQGGVLPNIQ AVLLPKK

UniProtKB: Histone H2A

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Macromolecule #2: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 10.979741 KDa
SequenceString:
KKRKRKESYA IYIYKVLKQV HPDTGISSKA MSIMNSFVND IFERIAAEAS RLAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKA VTKYTSSK

UniProtKB: Histone H2B

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Macromolecule #3: Histone H3

MacromoleculeName: Histone H3 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 11.74677 KDa
SequenceString:
KKPHRYRPGT VALREIRRYQ KSTELLIRKL PFQRLVREIA QDFKTDLRFQ SSAVMALQEA SEAYLVGLFE DTNLCAIHAK RVTIMPKDI QLARRIRGER A

UniProtKB: Histone H3

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Macromolecule #4: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 9.279875 KDa
SequenceString:
VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF GG

UniProtKB: Histone H4

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Macromolecule #5: DNA (164-MER)

MacromoleculeName: DNA (164-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 50.381172 KDa
SequenceString: (DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(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) ...String:
(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(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)(DG)(DA)(DT) (DA)(DT) (DA)(DT)

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Macromolecule #6: DNA (164-MER)

MacromoleculeName: DNA (164-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 50.861461 KDa
SequenceString: (DA)(DT)(DA)(DT)(DA)(DT)(DC)(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) ...String:
(DA)(DT)(DA)(DT)(DA)(DT)(DC)(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)(DG)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)(DA) (DT)(DA)

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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

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
10.0 mMHEPES-HClHEPES
5.0 percentGlycerolGlycerol
1.0 mMEDTAEDTA
350.0 ug/mLFLAGFLAG peptide

Details: 10 mM HEPES-HCl (pH = 7.5), 150 mM NaCl, 5% glycerol, 1 mM EDTA, 350 ug/mL 3x FLAG peptide, 1/1000th protease inhibitor
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
Detailsdouble FLAG-tagged Pol II subunit Rpb1 was used for purification of Pol II complexes. We aimed to purify Pol II in tandem with nucleosomes. The free nucleosomes were co-purified with this sample

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 31103 / Average electron dose: 50.65 e/Å2
Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was ...Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was achieved by extracting particles in box size 440x440 and Fourier-cropping it to box size 360x360
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1848096
Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (1,848,096) is the starting number of particles after initial cleanup and 3D ...Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (1,848,096) is the starting number of particles after initial cleanup and 3D classification of the data.
Startup modelType of model: INSILICO MODEL / In silico model: 'Ab initio' function in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1)
Details: The data was processed using cryoSPARC to 3.34 A from 319,428 particles. It was later post-processed using CryoSieve to select the final number of particles, yielding 3.29 A.
Number images used: 66988
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1) / Details: 'Non-Uniform Refinement' in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3lz0

source_name: PDB, initial_model_type: experimental model

2299

chain_id: a, source_name: AlphaFold, initial_model_type: in silico model

4040

chain_id: b, source_name: AlphaFold, initial_model_type: in silico model

4051

chain_id: c, source_name: AlphaFold, initial_model_type: in silico model

2283

chain_id: d, source_name: AlphaFold, initial_model_type: in silico model

2299

chain_id: e, source_name: AlphaFold, initial_model_type: in silico model

4040

chain_id: f, source_name: AlphaFold, initial_model_type: in silico model

4051

chain_id: g, source_name: AlphaFold, initial_model_type: in silico model

2283

chain_id: h, source_name: AlphaFold, initial_model_type: in silico model
DetailsThe model was based on PDB: 3LZ0. First, 3LZ0 was rigid-body fit in the cryo-EM Coulomb potential density map using UCSF ChimeraX. Next, AlphaFold2 models for individual Drosophila melanogaster histones were aligned to their 3LZ0 counterparts, and further optimized in the density using rigid-body fit. Then, in Coot, the models were Real-space refined in conjunction with DNA 601 Widom sequence. The DNA sequence was extended, to fit into the density. For the final model optimization, phenix.real_space_refine was used
RefinementProtocol: AB INITIO MODEL / Overall B value: 50
Output model

PDB-9mu4:
Structure of a native Drosophila melanogaster octameric nucleosome

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