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- PDB-6buz: Cryo-EM structure of CENP-A nucleosome in complex with kinetochor... -

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Entry
Database: PDB / ID: 6buz
TitleCryo-EM structure of CENP-A nucleosome in complex with kinetochore protein CENP-N
Components
  • (DNA (147-MER)) x 2
  • Histone H2A
  • Histone H2B
  • Histone H3-like centromeric protein A
  • Histone H4
  • Maltose-binding periplasmic protein, Centromere protein N chimera
KeywordsSTRUCTURAL PROTEIN/DNA / STRUCTURAL PROTEIN-DNA complex / Histone fold / Centromeric nucleosome / Kinetochore
Function/homologyKinetochore protein CHL4 like / Centromere protein Chl4/mis15/CENP-N / condensed chromosome inner kinetochore / kinetochore assembly / nuclear pericentric heterochromatin / protein localization to chromosome, centromeric region / condensed nuclear chromosome kinetochore / negative regulation of megakaryocyte differentiation / establishment of mitotic spindle orientation / CENP-A containing nucleosome assembly ...Kinetochore protein CHL4 like / Centromere protein Chl4/mis15/CENP-N / condensed chromosome inner kinetochore / kinetochore assembly / nuclear pericentric heterochromatin / protein localization to chromosome, centromeric region / condensed nuclear chromosome kinetochore / negative regulation of megakaryocyte differentiation / establishment of mitotic spindle orientation / CENP-A containing nucleosome assembly / Packaging Of Telomere Ends / Histone H2B signature. / Deposition of new CENPA-containing nucleosomes at the centromere / Histone H2B / condensed nuclear chromosome, centromeric region / Histone H2A signature. / Histone H2A conserved site / DNA replication-independent nucleosome assembly / Histone H2A, C-terminal domain / Histone H2A / chromatin silencing / telomere capping / condensed chromosome kinetochore / TATA box binding protein associated factor (TAF) / negative regulation of tumor necrosis factor-mediated signaling pathway / chromosome, centromeric region / Meiotic synapsis / Histone H4 signature. / carbohydrate transmembrane transporter activity / Histone H4 / Histone H4, conserved site / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / CENP-T/Histone H4, histone fold / innate immune response in mucosa / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / mitotic cytokinesis / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / SUMOylation of chromatin organization proteins / Mitotic Prometaphase / beta-catenin-TCF complex assembly / telomere organization / RNA Polymerase I Promoter Opening / DNA replication-dependent nucleosome assembly / C-terminus of histone H2A / SIRT1 negatively regulates rRNA expression / DNA methylation / Resolution of Sister Chromatid Cohesion / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / RNA Polymerase I Chain Elongation / Bacterial extracellular solute-binding protein / sister chromatid cohesion / nucleosomal DNA binding / PRC2 methylates histones and DNA / nuclear nucleosome / negative regulation of gene expression, epigenetic / NoRC negatively regulates rRNA expression / Condensation of Prophase Chromosomes / positive regulation of gene expression, epigenetic / Bacterial extracellular solute-binding protein / Nonhomologous End-Joining (NHEJ) / DNA-templated transcription, initiation / chromatin silencing at rDNA / Centromere kinetochore component CENP-T histone fold / HDACs deacetylate histones / Histone H3 signature 2. / Histone H3/CENP-A / nuclear chromosome / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / regulation of gene silencing by miRNA / Metalloprotease DUBs / RMTs methylate histone arginines / RHO GTPases Activate Formins / Histone H2A/H2B/H3 / regulation of megakaryocyte differentiation / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / double-strand break repair via nonhomologous end joining / nucleosome / PKMTs methylate histone lysines / Histone-fold / UCH proteinases / G2/M DNA damage checkpoint / DNA Damage/Telomere Stress Induced Senescence / lipopolysaccharide binding / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / nucleosome assembly / protein heterotetramerization / HDMs demethylate histones / Meiotic recombination / E3 ubiquitin ligases ubiquitinate target proteins / Separation of Sister Chromatids / Core histone H2A/H2B/H3/H4 / RUNX1 regulates transcription of genes involved in differentiation of HSCs / regulation of hematopoietic stem cell differentiation / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / nuclear chromosome, telomeric region / histone binding
Function and homology information
Specimen sourceHomo sapiens / / human
Escherichia coli / / bacteria /
Synthetic construct /
MethodElectron microscopy (3.92 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsChittori, S. / Hong, J. / Kelly, A.E. / Bai, Y. / Subramaniam, S.
CitationJournal: Science / Year: 2018
Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N.
Authors: Sagar Chittori / Jingjun Hong / Hayden Saunders / Hanqiao Feng / Rodolfo Ghirlando / Alexander E Kelly / Yawen Bai / Sriram Subramaniam
Abstract: Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by ...Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. We report cryo-electron microscopy (cryo-EM), biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We show that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrate analogous interactions in , which are further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with the coevolution of CENP-N and CENP-A and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.
Copyright: 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 11, 2017 / Release: Dec 20, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 20, 2017Structure modelrepositoryInitial release
1.1Jan 10, 2018Structure modelDatabase referencescitation_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
1.2Jan 31, 2018Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A
D: Histone H2B
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A
H: Histone H2B
I: DNA (147-MER)
J: DNA (147-MER)
N: Maltose-binding periplasmic protein, Centromere protein N chimera


Theoretical massNumber of molelcules
Total (without water)281,73711
Polyers281,73711
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Sedimentation Velocity
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)54640
ΔGint (kcal/M)-395
Surface area (Å2)84880

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Components

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Protein/peptide , 5 types, 9 molecules AEBFCGDHN

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


Mass: 18195.002 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens / Gene: CENPA / Production host: Escherichia coli / References: UniProt:P49450
#2: Protein/peptide Histone H4 /


Mass: 11394.426 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens
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 / References: UniProt:P62805
#3: Protein/peptide Histone H2A / / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14165.551 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens / Gene: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli / References: UniProt:P04908
#4: Protein/peptide Histone H2B / / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 13935.239 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens / Gene: HIST1H2BJ, H2BFR / Production host: Escherichia coli / References: UniProt:P06899
#7: Protein/peptide Maltose-binding periplasmic protein, Centromere protein N chimera / MBP / MMBP / Maltodextrin-binding protein / CENP-N / Interphase centromere complex protein 32


Mass: 75607.797 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli, (gene. exp.) Homo sapiens
Gene: malE, Z5632, ECs5017, CENPN, C16orf60, ICEN32, BM-309 / Production host: Escherichia coli / References: UniProt:P0AEY0, UniProt:Q96H22

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct
#6: DNA chain DNA (147-MER)


Mass: 45610.043 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Centromeric nucleosome in complex with kinetochore protein CENP-N
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7.4
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 kelvins

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Electron microscopy imaging

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

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFINDCTF CORRECTION
7UCSF ChimeraMODEL FITTING
12RELION2.0RECONSTRUCTION
19PHENIXMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 61749 / Symmetry type: POINT
Atomic model buildingDetails: Ab initio modeling of CENP-N backbone trace / Ref protocol: OTHER / Ref space: REAL / Target criteria: Model to Map correlation, Molprobity
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00813311
ELECTRON MICROSCOPYf_angle_d0.85819246
ELECTRON MICROSCOPYf_dihedral_angle_d24.3466952
ELECTRON MICROSCOPYf_chiral_restr0.0472216
ELECTRON MICROSCOPYf_plane_restr0.0051438

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