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

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Basic information

Entry
Database: PDB / ID: 9mu4
TitleStructure of a native Drosophila melanogaster octameric nucleosome
Components
  • (DNA (164-MER)) x 2
  • Histone H2A
  • Histone H2B
  • Histone H3
  • Histone H4
KeywordsGENE REGULATION / Nucleosome / histones / histone / chromatin / DNA
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 / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / HDACs deacetylate histones / Ub-specific processing proteases / RNA Polymerase I Promoter Escape / 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 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Senescence-Associated Secretory Phenotype (SASP) / Transcriptional regulation by small RNAs / Estrogen-dependent gene expression / 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 / structural constituent of chromatin / nucleosome / heterochromatin formation / nucleosome assembly / chromatin organization / chromosome / protein heterodimerization activity / protein-containing complex binding / chromatin / DNA binding / nucleus
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 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 / 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
DNA / DNA (> 10) / DNA (> 100) / Histone H2B / Histone H3 / Histone H4 / Histone H2A
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsVenette-Smith, N.L. / Vishwakarma, R.K. / Dollinger, R. / Schultz, J. / Venkatakrishnan, V. / Babitzke, P. / Anand, G. / Gilmour, D.S. / Armache, J.-P. / Murakami, K.
Funding support United States, 3items
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, N.L. / Vishwakarma, R.K. / Dollinger, R. / Schultz, J. / Venkatakrishnan, V. / Babitzke, P. / Anand, G. / Gilmour, D.S. / Armache, J.-P. / Murakami, K.
History
DepositionJan 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
c: Histone H2A
g: Histone H2A
d: Histone H2B
h: Histone H2B
a: Histone H3
e: Histone H3
b: Histone H4
f: Histone H4
T: DNA (164-MER)
N: DNA (164-MER)


Theoretical massNumber of molelcules
Total (without water)188,36010
Polymers188,36010
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 8 molecules cgdhaebf

#1: Protein Histone H2A


Mass: 11552.494 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: P84051
#2: Protein Histone H2B


Mass: 10979.741 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: P02283
#3: Protein Histone H3


Mass: 11746.770 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: P02299
#4: Protein Histone H4


Mass: 9279.875 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: P84040

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DNA chain , 2 types, 2 molecules TN

#5: DNA chain DNA (164-MER)


Mass: 50381.172 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly)
#6: DNA chain DNA (164-MER)


Mass: 50861.461 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly)

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Details

Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Natively purified octameric nucleosome from Drosophila melanogaster
Type: COMPLEX
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
Entity ID: all / Source: NATURAL
Molecular weightValue: 240 kDa/nm / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Buffer solutionpH: 7.5
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
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
210 mMHEPESHEPES-HCl1
35 percentGlycerolGlycerol1
41 mMEDTAEDTA1
5350 ug/mLFLAG peptideFLAG1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: double 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
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.65 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 31103
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
Image scansSampling size: 0.944 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.1particle selection
2EPU3.4image acquisition
4cryoSPARC4.3.1CTF correction
7UCSF ChimeraX1.8model fitting
9Coot0.9.8.7model refinement
10PHENIX1.21.5207model refinement
11cryoSPARC4.3.1initial Euler assignment
12cryoSPARC4.3.1final Euler assignment
14cryoSPARC4.3.13D reconstruction
CTF correctionDetails: 'Patch CTF Estimation' in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66988 / Algorithm: BACK PROJECTION
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.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 50 / Protocol: AB INITIO MODEL
Details: The 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 ...Details: The 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
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeChain-ID
13LZ0

3lz0

3LZ01PDBexperimental model
2P022992AlphaFoldin silico modela
3P840403AlphaFoldin silico modelb
4P840514AlphaFoldin silico modelc
5P022835AlphaFoldin silico modeld
6P022992AlphaFoldin silico modele
7P840403AlphaFoldin silico modelf
8P840514AlphaFoldin silico modelg
9P022835AlphaFoldin silico modelh
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00613690
ELECTRON MICROSCOPYf_angle_d0.8319900
ELECTRON MICROSCOPYf_dihedral_angle_d31.7914484
ELECTRON MICROSCOPYf_chiral_restr0.0472264
ELECTRON MICROSCOPYf_plane_restr0.0071388

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