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- EMDB-4711: Structure of LSD2/NPAC-linker/nucleosome core particle complex: C... -

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Basic information

Entry
Database: EMDB / ID: EMD-4711
TitleStructure of LSD2/NPAC-linker/nucleosome core particle complex: Class 4
Map dataLSD/NPAC(214-225)/nucleosome: class 4
Sample
  • Complex: LSD2/NPAC(214-225)/nucleosome
    • Protein or peptide: LSD2
    • Protein or peptide: NPAC
    • Protein or peptide: H3
    • Protein or peptide: H4
    • Protein or peptide: H2A
    • Protein or peptide: H2B
Biological speciesXenopus laevis (African clawed frog) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.23 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: 0.0105
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0105
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4711.png

Downloads & links

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Map

FileDownload / File: emd_4711.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLSD/NPAC(214-225)/nucleosome: class 4
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0105 / Movie #1: 0.0105
Minimum - Maximum-0.015342692 - 0.06420064
Average (Standard dev.)0.00026510516 (±0.0027025978)
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-0.0150.0640.000

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Supplemental data

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Sample components

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Entire : LSD2/NPAC(214-225)/nucleosome

EntireName: LSD2/NPAC(214-225)/nucleosome
Components
  • Complex: LSD2/NPAC(214-225)/nucleosome
    • Protein or peptide: LSD2
    • Protein or peptide: NPAC
    • Protein or peptide: H3
    • Protein or peptide: H4
    • Protein or peptide: H2A
    • Protein or peptide: H2B

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Supramolecule #1: LSD2/NPAC(214-225)/nucleosome

SupramoleculeName: LSD2/NPAC(214-225)/nucleosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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 weightExperimental: 290 KDa

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Macromolecule #1: LSD2

MacromoleculeName: LSD2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: PLGSRKCEKA GCTATCPVCF ASASERCAKN GYTSRWYHLS CGEHFCNECF DHYYRSHKDG YDKYTTWKKI WTSNGKTEPS PKAFMADQQ LPYWVQCTKP ECRKWRQLTK EIQLTPQIAK TYRCGMKPNT AIKPETSDHC SLPEDLRVLE VSNHWWYSML I LPPLLKDS ...String:
PLGSRKCEKA GCTATCPVCF ASASERCAKN GYTSRWYHLS CGEHFCNECF DHYYRSHKDG YDKYTTWKKI WTSNGKTEPS PKAFMADQQ LPYWVQCTKP ECRKWRQLTK EIQLTPQIAK TYRCGMKPNT AIKPETSDHC SLPEDLRVLE VSNHWWYSML I LPPLLKDS VAAPLLSAYY PDCVGMSPSC TSTNRAAATG NASPGKLEHS KAALSVHVPG MNRYFQPFYQ PNECGKALCV RP DVMELDE LYEFPEYSRD PTMYLALRNL ILALWYTNCK EALTPQKCIP HIIVRGLVRI RCVQEVERIL YFMTRKGLIN TGV LSVGAD QYLLPKDYHN KSVIIIGAGP AGLAAARQLH NFGIKVTVLE AKDRIGGRVW DDKSFKGVTV GRGAQIVNGC INNP VALMC EQLGISMHKF GERCDLIQEG GRITDPTIDK RMDFHFNALL DVVSEWRKDK TQLQDVPLGE KIEEIYKAFI KESGI QFSE LEGQVLQFHL SNLEYACGSN LHQVSARSWD HNEFFAQFAG DHTLLTPGYS VIIEKLAEGL DIQLKSPVQC IDYSGD EVQ VTTTDGTGYS AQKVLVTVPL ALLQKGAIQF NPPLSEKKMK AINSLGAGII EKIALQFPYR FWDSKVQGAD FFGHVPP SA SKRGLFAVFY DMDPQKKHSV LMSVIAGEAV ASVRTLDDKQ VLQQCMATLR ELFKEQEVPD PTKYFVTRWS TDPWIQMA Y SFVKTGGSGE AYDIIAEDIQ GTVFFAGEAT NRHFPQTVTG AYLSGVREAS KIAAF

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Macromolecule #2: NPAC

MacromoleculeName: NPAC / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
DPHFHHFLLS QT

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Macromolecule #3: H3

MacromoleculeName: H3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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 #4: H4

MacromoleculeName: H4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRD AVTYTEHAKR KTVTAMDVVY ALKRQGRTLY GFGG

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Macromolecule #5: H2A

MacromoleculeName: H2A / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK TRAKAKTRSS RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YLAAVLEYLT AEILELAGN AARDNKKTRI IPRHLQLAVR NDEELNKLLG GVTIAQGGVL PNIQSVLLPK K TESAKSAK SK

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Macromolecule #6: H2B

MacromoleculeName: H2B / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AKSAPAPKKG SKKAVTKTQK KDGKKRRKTR KESYAIYVYK VLKQVHPDTG ISSKAMSIMN SFVNDVFERI AGEASRLAHY NKRSTITSR EIQTAVRLLL PGELAKHAVS EGTKAVTKYT SAK

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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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 13798
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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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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