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- PDB-6r1u: Structure of LSD2/NPAC-linker/nucleosome core particle complex: C... -

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Basic information

Entry
Database: PDB / ID: 6r1u
TitleStructure of LSD2/NPAC-linker/nucleosome core particle complex: Class 2
Components
  • (DNA (147-MER)) x 2
  • Histone H2A
  • Histone H2B 1.1
  • Histone H3
  • Histone H3.2
  • Histone H4
  • Lysine-specific histone demethylase 1B
  • Putative oxidoreductase GLYR1
KeywordsGENE REGULATION / Histone demethylation / chromatin reader / flavoenzyme / epigenetics / evolution of protein function / molecular recognition.
Function / homology
Function and homology information


epigenetic programing of female pronucleus / genomic imprinting / [histone H3]-N6,N6-dimethyl-L-lysine4 FAD-dependent demethylase / FAD-dependent H3K4me/H3K4me3 demethylase activity / chromatin-protein adaptor activity / transcription elongation-coupled chromatin remodeling / nucleosome binding / histone demethylase activity / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / methylated histone binding ...epigenetic programing of female pronucleus / genomic imprinting / [histone H3]-N6,N6-dimethyl-L-lysine4 FAD-dependent demethylase / FAD-dependent H3K4me/H3K4me3 demethylase activity / chromatin-protein adaptor activity / transcription elongation-coupled chromatin remodeling / nucleosome binding / histone demethylase activity / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / methylated histone binding / transcription initiation-coupled chromatin remodeling / FAD binding / HDMs demethylate histones / structural constituent of chromatin / UCH proteinases / NAD binding / nucleosome / nucleosome assembly / flavin adenine dinucleotide binding / NADP binding / histone binding / oxidoreductase activity / chromatin remodeling / protein heterodimerization activity / chromatin binding / chromatin / DNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
NP60, PWWP domain / Zinc finger, CW-type / CW-type Zinc Finger / Zinc finger CW-type profile. / 3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain / NAD-binding of NADP-dependent 3-hydroxyisobutyrate dehydrogenase / : / 6-phosphogluconate dehydrogenase, NADP-binding / NAD binding domain of 6-phosphogluconate dehydrogenase / SWIRM domain ...NP60, PWWP domain / Zinc finger, CW-type / CW-type Zinc Finger / Zinc finger CW-type profile. / 3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain / NAD-binding of NADP-dependent 3-hydroxyisobutyrate dehydrogenase / : / 6-phosphogluconate dehydrogenase, NADP-binding / NAD binding domain of 6-phosphogluconate dehydrogenase / SWIRM domain / SWIRM domain / SWIRM domain profile. / : / 6-phosphogluconate dehydrogenase, domain 2 / Amine oxidase / Flavin containing amine oxidoreductase / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP domain / 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 / Homeobox-like domain superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / FAD/NAD(P)-binding domain superfamily / Histone-fold / NAD(P)-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / DNA / DNA (> 10) / DNA (> 100) / Histone H3 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Cytokine-like nuclear factor N-PAC ...FLAVIN-ADENINE DINUCLEOTIDE / DNA / DNA (> 10) / DNA (> 100) / Histone H3 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Cytokine-like nuclear factor N-PAC / Histone H2A / Lysine-specific histone demethylase 2
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.36 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
Revision 1.1Oct 30, 2019Group: Data collection / Source and taxonomy / Structure summary
Category: entity / entity_src_gen / pdbx_entity_src_syn / Item: _entity.src_method
Revision 1.2May 15, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • EMDB-4705
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A
D: Histone H2B 1.1
E: Histone H3.2
F: Histone H4
G: Histone H2A
H: Histone H2B 1.1
I: DNA (147-MER)
J: DNA (147-MER)
K: Lysine-specific histone demethylase 1B
L: Putative oxidoreductase GLYR1
M: Histone H3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)315,75717
Polymers314,77513
Non-polymers9824
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area62950 Å2
ΔGint-448 kcal/mol
Surface area108180 Å2
MethodPISA

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Components

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Protein , 7 types, 11 molecules AEBFCGDHKLM

#1: Protein Histone H3.2


Mass: 15303.930 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) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: P84233
#2: Protein Histone H4


Mass: 11263.231 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) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: P62799
#3: Protein Histone H2A


Mass: 13978.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, LOC494591 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13524.752 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) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: P02281
#7: Protein Lysine-specific histone demethylase 1B / Flavin-containing amine oxidase domain-containing protein 1 / Lysine-specific histone demethylase 2


Mass: 87051.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KDM1B, AOF1, C6orf193, LSD2 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: Q8NB78, Oxidoreductases
#8: Protein Putative oxidoreductase GLYR1 / 3-hydroxyisobutyrate dehydrogenase-like protein / Cytokine-like nuclear factor N-PAC / Glyoxylate ...3-hydroxyisobutyrate dehydrogenase-like protein / Cytokine-like nuclear factor N-PAC / Glyoxylate reductase 1 homolog / Nuclear protein NP60 / Nuclear protein of 60 kDa


Mass: 13528.271 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GLYR1, HIBDL, NP60 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q49A26, Oxidoreductases
#9: Protein Histone H3


Mass: 15305.945 Da / Num. of mol.: 1
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) / Strain (production host): BL21 / Variant (production host): pLysS / References: UniProt: A0A310TTQ1, UniProt: P84233*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: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 4 molecules

#10: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C27H33N9O15P2 / Comment: FAD*YM
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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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
1LSD2/NPAC(214-225)/nucleosomeCOMPLEXXenopus laevis histones recombinantly expressed. Alkylated K4C-C110A H3. 601 Widom DNA sequence. Human LSD2 Human NPAC#1-#90MULTIPLE SOURCES
2HistoneCOMPLEX#1-#4, #91RECOMBINANT
3DNACOMPLEX#5-#61RECOMBINANT
4Lysine-specific histone demethylase 1BCOMPLEX#71RECOMBINANT
5Putative oxidoreductase GLYR1COMPLEX#81RECOMBINANT
Molecular weightValue: 0.29 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Xenopus laevis (African clawed frog)8355
33synthetic construct (others)32630
44Homo sapiens (human)9606
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)511693
33synthetic construct (others)32630
44Escherichia coli BL21 (bacteria)511693
55synthetic 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.
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 FIELD / 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
IDNameVersionCategoryFitting-ID
1RELION2.1particle selection
7UCSF Chimeramodel fitting1
12RELION33D reconstruction
20UCSF Chimeramodel fitting2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 490558
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72315 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1RIGID BODY FITREAL
2RIGID BODY FITREAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16ESF

6esf

16ESF1PDBexperimental model
24HSU

4hsu

24HSU2PDBexperimental model

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