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- PDB-6e0p: Cryo-EM structure of the centromeric nucleosome (Native alpha sat... -

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Entry
Database: PDB / ID: 6e0p
TitleCryo-EM structure of the centromeric nucleosome (Native alpha satellite DNA) in complex with a single chain antibody fragment
Components
  • (DNA (145-MER)) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3-like centromeric protein A
  • Histone H4
  • scFvSingle-chain variable fragment
KeywordsNUCLEAR PROTEIN / CENP-A / Centromere / Native alpha satellite DNA / anti-nucleosome antibody / acidic patch / nucleosome
Function / homology
Function and homology information


Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / Formation of the beta-catenin:TCF transactivating complex / Pre-NOTCH Transcription and Translation / Packaging Of Telomere Ends ...Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / Formation of the beta-catenin:TCF transactivating complex / Pre-NOTCH Transcription and Translation / Packaging Of Telomere Ends / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Meiotic synapsis / Cleavage of the damaged purine / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged pyrimidine / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / PKMTs methylate histone lysines / HDMs demethylate histones / Amyloid fiber formation / Meiotic recombination / Estrogen-dependent gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / RNA Polymerase I Promoter Escape / RNA Polymerase I Promoter Opening / G2/M DNA damage checkpoint / Mitotic Prometaphase / Deposition of new CENPA-containing nucleosomes at the centromere / Processing of DNA double-strand break ends / Nonhomologous End-Joining (NHEJ) / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Metalloprotease DUBs / Ub-specific processing proteases / SUMOylation of chromatin organization proteins / HATs acetylate histones / RMTs methylate histone arginines / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / NoRC negatively regulates rRNA expression / B-WICH complex positively regulates rRNA expression / DNA methylation / Transcriptional regulation by small RNAs / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RHO GTPases Activate Formins / UCH proteinases / condensed chromosome inner kinetochore / kinetochore assembly / protein localization to chromosome, centromeric region / nuclear pericentric heterochromatin / condensed nuclear chromosome kinetochore / establishment of mitotic spindle orientation / negative regulation of megakaryocyte differentiation / condensed nuclear chromosome, centromeric region / CENP-A containing nucleosome assembly / chromosome, centromeric region / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / mitotic cytokinesis / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / nuclear nucleosome / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / nucleosomal DNA binding / DNA-templated transcription, initiation / regulation of gene silencing by miRNA / nuclear chromosome / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / lipopolysaccharide binding / protein heterotetramerization / chromatin organization / double-strand break repair via nonhomologous end joining / antibacterial humoral response / nuclear chromosome, telomeric region / killing of cells of other organism / antimicrobial humoral immune response mediated by antimicrobial peptide / nuclear chromatin / protein ubiquitination / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / protein domain specific binding / chromatin binding / viral process / cellular protein metabolic process / protein heterodimerization activity / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / nucleoplasm
Histone H4, conserved site / Histone H3/CENP-A / Histone H2B signature. / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone-fold / Histone H3 signature 2. ...Histone H4, conserved site / Histone H3/CENP-A / Histone H2B signature. / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone-fold / Histone H3 signature 2. / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / Centromere kinetochore component CENP-T histone fold / C-terminus of histone H2A / Histone H2A signature. / Histone H4 signature.
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H3-like centromeric protein A / Histone H4
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsYadav, K.N.S. / Zhou, B.-R.
Funding supportUnited States , 1件
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institutethe intramural 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 6, 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

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (145-MER)
J: DNA (145-MER)
M: scFv
N: scFv


Theoretical massNumber of molelcules
Total (without water)262,61712
Polymers262,61712
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 5 types, 10 molecules AEBFCGDHMN

#1: Protein/peptide Histone H3-like centromeric protein A / Centromere autoantigen A / Centromere protein A / CENP-A


Mass: 18038.818 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPA / Production host: Escherichia coli (E. coli) / References: UniProt: P49450
#2: Protein/peptide Histone H4 /


Mass: 11394.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein/peptide Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14165.551 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein/peptide Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 13935.239 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BJ, H2BFR / Production host: Escherichia coli (E. coli) / References: UniProt: P06899
#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 (145-MER)


Mass: 44539.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (145-MER)


Mass: 44948.793 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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: CENP-A nucleosome and antibody complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Details: blot for 2.5 sec before plunging

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 15.2 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1416
Details: Images were collected in movie-mode at 38 frames over 15.2 seconds
Image scansMovie frames/image: 38 / Used frames/image: 1-38

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
7UCSF Chimera1.13.1model fitting
10RELION3.0 beta2final Euler assignment
11RELION3.0 betaclassification
12RELION3.0 beta23D reconstruction
13PHENIX1.13-2998model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 301644 / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3AN2

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