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- PDB-6r1t: Structure of LSD2/NPAC-linker/nucleosome core particle complex: C... -

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Basic information

Entry
Database: PDB / ID: 6r1t
TitleStructure of LSD2/NPAC-linker/nucleosome core particle complex: Class 1, free nuclesome
Components
  • (DNA (147-MER)) x 2
  • HISTONE H2A
  • Histone H2A
  • Histone H2B
  • Histone H3
KeywordsGENE REGULATION / Histone demethylation / chromatin reader / flavoenzyme / epigenetics / evolution of protein function / molecular recognition.
Function / homology
Function and homology information


structural constituent of chromatin / nucleosome / protein heterodimerization activity / DNA binding / nucleoplasm / nucleus
Similarity search - Function
Histone, subunit A / Histone, subunit A / 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, subunit A / Histone, subunit A / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2B / Histone H3 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Histone H2A
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsMarabelli, C. / Pilotto, S. / Chittori, S. / Subramaniam, S. / Mattevi, A.
Funding support Italy, 1items
OrganizationGrant numberCountry
Italian Association for Cancer ResearchIG-11342 Italy
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • EMDB-4704
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3
B: HISTONE H2A
C: Histone H2A
D: Histone H2B
E: Histone H3
F: HISTONE H2A
G: Histone H2A
H: Histone H2B
I: DNA (147-MER)
J: DNA (147-MER)


Theoretical massNumber of molelcules
Total (without water)179,94510
Polymers179,94510
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56470 Å2
ΔGint-425 kcal/mol
Surface area77140 Å2
MethodPISA

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3 /


Mass: 11631.616 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: XELAEV_18002543mg / Production host: Escherichia coli (E. coli) / References: UniProt: A0A310TTQ1, UniProt: P84233*PLUS
#2: Protein HISTONE H2A /


Mass: 9990.770 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P62799*PLUS
#3: Protein Histone H2A /


Mass: 12183.232 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, LOC494591, XELAEV_18003602mg / Production host: Escherichia coli (E. coli) / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#4: Protein Histone H2B /


Mass: 10792.419 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: XELAEV_18032686mg / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1L8FQ56, UniProt: P02281*PLUS

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (147-MER)


Mass: 45610.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#6: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: 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
IDNameTypeDetailsEntity IDParent-IDSource
1147 bp nucleosomeCOMPLEXXenopus laevis histones recombinantly expressed. Alkylated K4C-C110A H3. 601 Widom DNA sequence.all0MULTIPLE SOURCES
2HistonesHistoneCOMPLEX#1-#41RECOMBINANT
3Widom DNACOMPLEX#5-#61RECOMBINANT
Molecular weightValue: 0.19866 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Xenopus laevis (African clawed frog)8355
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
210 mMPotassium ChlorideKCl1
SpecimenConc.: 0.87 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: LSD2/NPAC(214-225)/nucleosome was monodisperse (gel filtration peak isolation and concentration in buffer described.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3.05 nm / Nominal defocus min: 0.7 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 1.25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2078
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
7UCSF Chimeramodel fitting
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 490558
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124354 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6ESF

6esf

RefinementHighest resolution: 3.7 Å / Cross valid method: THROUGHOUT

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