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- PDB-7wlr: Cryo-EM structure of the nucleosome containing Komagataella pasto... -

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Basic information

Entry
Database: PDB / ID: 7wlr
TitleCryo-EM structure of the nucleosome containing Komagataella pastoris histones
Components
  • (DNA (145-MER)) x 2
  • Histone H2A
  • Histone H2B
  • Histone H3
  • Histone H4
KeywordsGENE REGULATION/DNA / Chromatin / Nucleosome / Komagataella pastoris / Histone H2A / Histone H2B / Histone H3 / Histone H4 / DNA / Epigenetic / Gene regulation / cryo-EM / GENE REGULATION-DNA complex
Function / homology
Function and homology information


rRNA transcription / structural constituent of chromatin / nucleosome / nucleosome assembly / protein heterodimerization activity / DNA repair / 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 H4, conserved site / Histone H4 signature. / Histone H4 ...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 H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / 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
DNA / DNA (> 10) / DNA (> 100) / Histone H4 / Histone H3 / Histone H2B / Histone H2A
Similarity search - Component
Biological speciesKomagataella pastoris (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsFukushima, Y. / Hatazawa, S. / Hirai, S. / Kujirai, T. / Takizawa, Y. / Kurumizaka, H.
Funding support Japan, 7items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 26, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 (145-MER)
J: DNA (145-MER)


Theoretical massNumber of molelcules
Total (without water)178,43510
Polymers178,43510
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3


Mass: 11601.565 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella pastoris (fungus) / Gene: ATY40_BA7502079, ATY40_BA7504318 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1B2JB78
#2: Protein Histone H4


Mass: 10030.811 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella pastoris (fungus) / Gene: ATY40_BA7502080, ATY40_BA7504319 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1B2JA70
#3: Protein Histone H2A


Mass: 11810.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella pastoris (fungus) / Gene: HTA1, ATY40_BA7502752 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1B2JD99
#4: Protein Histone H2B


Mass: 11018.554 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella pastoris (fungus) / Gene: ATY40_BA7502753 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1B2JBS1

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-MER)


Mass: 44520.383 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (145-MER)


Mass: 44991.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1K. pastoris nucleosomeCOMPLEX#1-#40MULTIPLE SOURCES
2HistoneCOMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#1-#21SYNTHETIC
Source (natural)Organism: Komagataella pastoris (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1250 nm
Image recordingElectron dose: 57 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: RELION / Version: 3.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181796 / Symmetry type: POINT

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