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- PDB-6dzt: Cryo-EM structure of nucleosome in complex with a single chain an... -

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Basic information

Entry
Database: PDB / ID: 6dzt
TitleCryo-EM structure of nucleosome in complex with a single chain antibody fragment
Components
  • (DNA (147-MER)) x 2
  • Histone H2A
  • Histone H2B
  • Histone H3
  • Histone H4
  • scFvSingle-chain variable fragment
KeywordsNUCLEAR PROTEIN / Nucleosome / Single chain antibody / charge-charge interaction / acidic patch.
Function / homology
Function and homology information


SUMOylation of chromatin organization proteins / Interleukin-7 signaling / PRC2 methylates histones and DNA / Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / Senescence-Associated Secretory Phenotype (SASP) / PKMTs methylate histone lysines / HDMs demethylate histones / HATs acetylate histones / RMTs methylate histone arginines ...SUMOylation of chromatin organization proteins / Interleukin-7 signaling / PRC2 methylates histones and DNA / Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / Senescence-Associated Secretory Phenotype (SASP) / PKMTs methylate histone lysines / HDMs demethylate histones / HATs acetylate histones / RMTs methylate histone arginines / SIRT1 negatively regulates rRNA expression / Factors involved in megakaryocyte development and platelet production / Estrogen-dependent gene expression / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / RNA Polymerase I Promoter Escape / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Chromatin modifying enzymes / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / Ub-specific processing proteases / Transcriptional regulation by small RNAs / RCAF complex / polytene chromosome band / larval somatic muscle development / polytene chromosome / chromatin assembly or disassembly / female meiosis chromosome segregation / nuclear nucleosome / nucleosomal DNA binding / DNA-templated transcription, initiation / nuclear chromosome / nucleosome assembly / nucleosome / chromosome / chromatin organization / histone binding / nuclear chromatin / protein-containing complex binding / protein heterodimerization activity / DNA binding / nucleus
Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H4, conserved site / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / C-terminus of histone H2A / Histone H2A signature. / Histone H4 signature. ...Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H4, conserved site / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / C-terminus of histone H2A / Histone H2A signature. / Histone H4 signature. / Histone H2B signature. / Histone H2A/H2B/H3 / Histone H3 signature 2. / Histone-fold / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H3/CENP-A / Histone H2B / Histone H4
Histone H2B / Histone H3 / Histone H4 / Histone H2A
Biological speciesDrosophila melanogaster (fruit fly)
Mus musculus (house mouse)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsYadav, K.N.S. / Zhou, B.-R.
Funding supportUnited States , 1件
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteIntramural Research ProgramUnited 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 5, 2018 / Release: May 22, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 22, 2019Structure modelrepositoryInitial release
1.1Jun 12, 2019Structure modelData collection / Database referencescitation / citation_author_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
1.2Jun 19, 2019Structure modelData collection / Database referencescitation_author_citation_author.identifier_ORCID

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3
B: Histone H4
C: Histone H2A
D: Histone H2B
E: Histone H3
F: Histone H4
G: Histone H2A
H: Histone H2B
I: DNA (147-MER)
J: DNA (147-MER)
M: scFv
N: scFv


Theoretical massNumber of molelcules
Total (without water)257,15212
Polymers257,15212
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein/peptide , 5 types, 10 molecules AEBFCGDHMN

#1: Protein/peptide Histone H3 /


Mass: 15421.101 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: His3, His3:CG31613, CG31613, His3:CG33803, CG33803, His3:CG33806, CG33806, His3:CG33809, CG33809, His3:CG33812, CG33812, His3:CG33815, CG33815, His3:CG33818, CG33818, His3:CG33821, CG33821, His3:CG33824, CG33824, His3:CG33827, CG33827, His3:CG33830, CG33830, His3:CG33833, CG33833, His3:CG33836, CG33836, His3:CG33839, CG33839, His3:CG33842, CG33842, His3:CG33845, CG33845, His3:CG33848, CG33848, His3:CG33851, CG33851, His3:CG33854, CG33854, His3:CG33857, CG33857, His3:CG33860, CG33860, His3:CG33863, CG33863, His3:CG33866, CG33866
Production host: Escherichia coli (E. coli) / References: UniProt: P02299
#2: Protein/peptide Histone H4 /


Mass: 11521.611 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: His4, H4, His4r, H4r, CG3379, His4:CG31611, CG31611, His4:CG33869, CG33869, His4:CG33871, CG33871, His4:CG33873, CG33873, His4:CG33875, CG33875, His4:CG33877, CG33877, His4:CG33879, CG33879, His4:CG33881, CG33881, His4:CG33883, CG33883, His4:CG33885, CG33885, His4:CG33887, CG33887, His4:CG33889, CG33889, His4:CG33891, CG33891, His4:CG33893, CG33893, His4:CG33895, CG33895, His4:CG33897, CG33897, His4:CG33899, CG33899, His4:CG33901, CG33901, His4:CG33903, CG33903, His4:CG33905, CG33905, His4:CG33907, CG33907, His4:CG33909, CG33909
Production host: Escherichia coli (E. coli) / References: UniProt: P84040
#3: Protein/peptide Histone H2A /


Mass: 13388.727 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: His2A, H2a, His2A:CG31618, CG31618, His2A:CG33808, CG33808, His2A:CG33814, CG33814, His2A:CG33817, CG33817, His2A:CG33820, CG33820, His2A:CG33823, CG33823, His2A:CG33826, CG33826, His2A:CG33829, CG33829, His2A:CG33832, CG33832, His2A:CG33835, CG33835, His2A:CG33838, CG33838, His2A:CG33841, CG33841, His2A:CG33844, CG33844, His2A:CG33847, CG33847, His2A:CG33850, CG33850, His2A:CG33862, CG33862, His2A:CG33865, CG33865
Production host: Escherichia coli (E. coli) / References: UniProt: P84051
#4: Protein/peptide Histone H2B /


Mass: 13840.224 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: His2B, His2B:CG17949, CG17949, His2B:CG33868, CG33868, His2B:CG33870, CG33870, His2B:CG33872, CG33872, His2B:CG33874, CG33874, His2B:CG33876, CG33876, His2B:CG33878, CG33878, His2B:CG33880, CG33880, His2B:CG33882, CG33882, His2B:CG33884, CG33884, His2B:CG33886, CG33886, His2B:CG33888, CG33888, His2B:CG33890, CG33890, His2B:CG33892, CG33892, His2B:CG33894, CG33894, His2B:CG33896, CG33896, His2B:CG33898, CG33898, His2B:CG33900, CG33900, His2B:CG33902, CG33902, His2B:CG33904, CG33904, His2B:CG33906, CG33906, His2B:CG33908, CG33908, His2B:CG33910, CG33910
Production host: Escherichia coli (E. coli) / References: UniProt: P02283
#7: Protein/peptide scFv / Single-chain variable fragment


Mass: 29030.146 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (147-MER)


Mass: 45610.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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: Nucleosome-Antibody complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7 / Source: RECOMBINANT
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.13_2998refinement
PHENIX1.13_2998refinement
EM softwareName: RELION / Version: 3.0 beta / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238790 / Num. of class averages: 2 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00816523
f_angle_d0.8823586
f_chiral_restr0.05162655
f_plane_restr0.00741956
f_dihedral_angle_d21.4388912

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