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- EMDB-32591: Cryo-EM structure of the nucleosome containing Komagataella pasto... -

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Basic information

Entry
Database: EMDB / ID: EMD-32591
TitleCryo-EM structure of the nucleosome containing Komagataella pastoris histones
Map dataCryo-EM map of the nucleosome containing Komagataella pastoris histones
Sample
  • Complex: K. pastoris nucleosome
    • Complex: Histone
      • Protein or peptide: Histone H3
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
    • Complex: DNA
  • DNA: DNA (145-MER)
  • DNA: DNA (145-MER)
Function / homology
Function and homology information


chromosome, centromeric region / DNA-templated transcription initiation / nucleosome / protein heterodimerization activity / 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 H4, conserved site ...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 H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / 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 H4 / Histone H3 / Histone H2B / Histone H2A
Similarity search - Component
Biological speciesKomagataella pastoris (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsFukushima Y / Hatazawa S / Hirai S / Kujirai T / Takizawa Y / Kurumizaka H
Funding support Japan, 7 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19K06522 Japan
Japan Society for the Promotion of Science (JSPS)JP20K15711 Japan
Japan Society for the Promotion of Science (JSPS)JP18H05534 Japan
Japan Society for the Promotion of Science (JSPS)JP20H00449 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Science and TechnologyJPMJCR16G1 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101076 Japan
CitationJournal: J Biochem / Year: 2022
Title: Structural and biochemical analyses of the nucleosome containing Komagataella pastoris histones.
Authors: Yutaro Fukushima / Suguru Hatazawa / Seiya Hirai / Tomoya Kujirai / Haruhiko Ehara / Shun-Ichi Sekine / Yoshimasa Takizawa / Hitoshi Kurumizaka /
Abstract: Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and ...Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and determined the structure of the nucleosome core particle by cryogenic electron microscopy. In the K. pastoris nucleosome, the histones form an octamer and the DNA is left-handedly wrapped around it. Micrococcal nuclease assays revealed that the DNA ends of the K. pastoris nucleosome are somewhat more accessible, as compared with those of the human nucleosome. In vitro transcription assays demonstrated that the K. pastoris nucleosome is transcribed by the K. pastoris RNA polymerase II (RNAPII) more efficiently than the human nucleosome, while the RNAPII pausing positions of the K. pastoris nucleosome are the same as those of the human nucleosome. These results suggested that the DNA end flexibility may enhance the transcription efficiency in the nucleosome but minimally affect the nucleosomal pausing positions of RNAPII.
History
DepositionJan 13, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateAug 10, 2022-
Current statusAug 10, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32591.png

Downloads & links

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Map

FileDownload / File: emd_32591.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the nucleosome containing Komagataella pastoris histones
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 3.94
Minimum - Maximum-16.197868 - 26.531948
Average (Standard dev.)6.5407666e-09 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 190.79999 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : K. pastoris nucleosome

EntireName: K. pastoris nucleosome
Components
  • Complex: K. pastoris nucleosome
    • Complex: Histone
      • Protein or peptide: Histone H3
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
    • Complex: DNA
  • DNA: DNA (145-MER)
  • DNA: DNA (145-MER)

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Supramolecule #1: K. pastoris nucleosome

SupramoleculeName: K. pastoris nucleosome / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Komagataella pastoris (fungus)

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Supramolecule #2: Histone

SupramoleculeName: Histone / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1-#4

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #1-#2

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Macromolecule #1: Histone H3

MacromoleculeName: Histone H3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 11.601565 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KPHRYKPGTV ALREIRRFQK STELLIRKLP FQRLVREIAQ DFKTDLRFQS SAIGALQESV EAYLVSLFED TNLCAIHAKR VTIQKKDIL LARRLRGERS

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 10.030811 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KRHRKILRDN IQGITKPAIR RLARRGGVKR ISALIYEEVR AVLKTFLENV IRDAVTYTEH AKRKTVTSLD VVYALKRQGR TLYGFGG

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Macromolecule #3: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 11.810682 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KASTSRSSKA GLTFPVGRVH RLLRRGNYAQ RIGSGAPVYL TAVLEYLAAE ILELAGNAAR DNKKSRIIPR HLQLAIRNDE ELNKLLGHV TIAQGGVLPN IQSELLPKK

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Macromolecule #4: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 11.018554 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
RSKVRKESYA SYIYKVLKQT HPDTGISQKA MSIMNSFVND IFERIASEAS KLASYNKKST ISAREIQTAV RLILPGELSK HAVSEGTRA VTKYTSSTQA

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Macromolecule #5: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.520383 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #6: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.99166 KDa
SequenceString: (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) ...String:
(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)

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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

Concentration1.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3lz0

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 181796
FSC plot (resolution estimation)

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