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- EMDB-4712: Structure of LSD2/NPAC-linker/nucleosome core particle complex: C... -

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Basic information

Entry
Database: EMDB / ID: EMD-4712
TitleStructure of LSD2/NPAC-linker/nucleosome core particle complex: Class 5
Map dataUnfolded nucleosome
Sample
  • Complex: partly unfolded nucleosome
    • Protein or peptide: H3
    • Protein or peptide: H4
    • Protein or peptide: H2A
    • Protein or peptide: H2B
    • DNA: DNA (147mer)
    • DNA: DNA (147mer)
    • Protein or peptide: H3
    • Protein or peptide: H4
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.95 Å
AuthorsMarabelli C / Pilotto S / Chittori S / Subramaniam S / Mattevi A
CitationJournal: Cell Rep / Year: 2019
Title: A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.
Authors: Chiara Marabelli / Biagina Marrocco / Simona Pilotto / Sagar Chittori / Sarah Picaud / Sara Marchese / Giuseppe Ciossani / Federico Forneris / Panagis Filippakopoulos / Guy Schoehn / Daniela ...Authors: Chiara Marabelli / Biagina Marrocco / Simona Pilotto / Sagar Chittori / Sarah Picaud / Sara Marchese / Giuseppe Ciossani / Federico Forneris / Panagis Filippakopoulos / Guy Schoehn / Daniela Rhodes / Sriram Subramaniam / Andrea Mattevi /
Abstract: LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase ...LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
History
DepositionMar 15, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 2019-
UpdateApr 24, 2019-
Current statusApr 24, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0081
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0081
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4712.png

Downloads & links

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Map

FileDownload / File: emd_4712.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnfolded nucleosome
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0081 / Movie #1: 0.0081
Minimum - Maximum-0.007963713 - 0.05830032
Average (Standard dev.)0.00018798585 (±0.0022271816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z318.000318.000318.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0080.0580.000

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Supplemental data

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Sample components

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Entire : partly unfolded nucleosome

EntireName: partly unfolded nucleosome
Components
  • Complex: partly unfolded nucleosome
    • Protein or peptide: H3
    • Protein or peptide: H4
    • Protein or peptide: H2A
    • Protein or peptide: H2B
    • DNA: DNA (147mer)
    • DNA: DNA (147mer)
    • Protein or peptide: H3
    • Protein or peptide: H4

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Supramolecule #1: partly unfolded nucleosome

SupramoleculeName: partly unfolded nucleosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: Xenopus laevis histones recombinantly expressed. Alkylated K4C-C110A H3. 601 Widom DNA sequence. Human LSD2 Human NPAC
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 178 KDa

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Macromolecule #1: H3

MacromoleculeName: H3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAV MALQEASEAY LVALFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA

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Macromolecule #2: H4

MacromoleculeName: H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #3: H2A

MacromoleculeName: H2A / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKQGGKT RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAVRN DEELNKLLGR VTIAQGGVLP NIQSVLLPKK TESSKSAKSK

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Macromolecule #4: H2B

MacromoleculeName: H2B / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPDPAKSAPA AKKGSKKAVT KTQKKDGKKR RKSRKESYAI YVYKVLKQVH PDTGISSKAM SIMNSFVND VFERIAGEAS RLAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV T KYTSAK

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Macromolecule #7: H3

MacromoleculeName: H3 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAV MALQEASEAY LVALFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA

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Macromolecule #8: H4

MacromoleculeName: H4 / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #5: DNA (147mer)

MacromoleculeName: DNA (147mer) / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATCGGATGTA TATATCTGAC ACGTGCCTGG AGACTAGGGA GTAATCCCCT TGGCGGTTAA AACGCGGGGG ACAGCGCGTA CGTGCGTTT AAGCGGTGCT AGAGCTGTCT ACGACCAATT GAGCGGCCTC GGCACCGGGA TTCTCGAT

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Macromolecule #6: DNA (147mer)

MacromoleculeName: DNA (147mer) / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATCGAGAATC CCGGTGCCGA GGCCGCTCAA TTGGTCGTAG ACAGCTCTAG CACCGCTTAA ACGCACGTAC GCGCTGTCCC CCGCGTTTT AACCGCCAAG GGGATTACTC CCTAGTCTCC AGGCACGTGT CAGATATATA CATCCGAT

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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

Concentration0.87 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
10.0 mMKClPotassium Chloride
VitrificationCryogen name: ETHANE
DetailsLSD2/NPAC(214-225)/nucleosome was monodisperse (gel filtration peak isolation and concentration in buffer described.

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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: 0.00305 µm / Nominal defocus min: 0.0007 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2078 / Average exposure time: 8.0 sec. / Average electron dose: 1.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 490558
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6esf

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 29748

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Atomic model buiding 1

Initial modelPDB ID:

6esf

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4hsu

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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