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- PDB-6nn6: Structure of Dot1L-H2BK120ub nucleosome complex -

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Basic information

Entry
Database: PDB / ID: 6nn6
TitleStructure of Dot1L-H2BK120ub nucleosome complex
Components
  • (DNA (145-MER)) x 2
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
  • Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
  • Ubiquitin
KeywordsGENE REGULATION / chromatin / structural biology / single particle / cryo-EM / histone methyltransferase / nucleosome / Dot1L
Function / homology
Function and homology information


: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / histone H3 methyltransferase activity / regulation of receptor signaling pathway via JAK-STAT / histone methyltransferase activity / heterochromatin formation / telomere organization ...: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / histone H3 methyltransferase activity / regulation of receptor signaling pathway via JAK-STAT / histone methyltransferase activity / heterochromatin formation / telomere organization / DNA damage checkpoint signaling / PKMTs methylate histone lysines / structural constituent of chromatin / nucleosome / gene expression / RNA polymerase II-specific DNA-binding transcription factor binding / nucleic acid binding / transcription coactivator activity / protein heterodimerization activity / DNA repair / intracellular membrane-bounded organelle / positive regulation of cell population proliferation / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal Domain) / Histone H2B signature. ...434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal 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 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 / Vaccinia Virus protein VP39 / Histone H3 signature 1. / Ubiquitin conserved site / Ubiquitin domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Ubiquitin domain signature. / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Ubiquitin C / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Histone-lysine N-methyltransferase, H3 lysine-79 specific
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsAnderson, C.J. / Baird, M.R. / Hsu, A. / Barbour, E.H. / Koyama, Y. / Borgnia, M.J. / McGinty, R.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)T32GM008570 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)ZIC ES103326 United States
CitationJournal: Cell Rep / Year: 2019
Title: Structural Basis for Recognition of Ubiquitylated Nucleosome by Dot1L Methyltransferase.
Authors: Cathy J Anderson / Matthew R Baird / Allen Hsu / Emily H Barbour / Yuka Koyama / Mario J Borgnia / Robert K McGinty /
Abstract: Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of ...Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of the nucleosome, is mediated by the Dot1L methyltransferase. Dot1L activity is part of a trans-histone crosstalk pathway, requiring prior histone H2B ubiquitylation of lysine 120 (H2BK120ub) for optimal activity. However, the molecular details describing both how Dot1L binds to the nucleosome and why Dot1L is activated by H2BK120 ubiquitylation are unknown. Here, we present the cryoelectron microscopy (cryo-EM) structure of Dot1L bound to a nucleosome reconstituted with site-specifically ubiquitylated H2BK120. The structure reveals that Dot1L engages the nucleosome acidic patch using a variant arginine anchor and occupies a conformation poised for methylation. In this conformation, Dot1L and ubiquitin interact directly through complementary hydrophobic surfaces. This study establishes a path to better understand Dot1L function in normal and leukemia cells.
History
DepositionJan 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 13, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 27, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 20, 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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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B 1.1
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B 1.1
I: DNA (145-MER)
J: DNA (145-MER)
K: Histone-lysine N-methyltransferase, H3 lysine-79 specific
L: Ubiquitin


Theoretical massNumber of molelcules
Total (without water)254,26212
Polymers254,26212
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
MethodPISA

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Components

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Protein , 6 types, 10 molecules AEBFCGDHKL

#1: Protein Histone H3.2


Mass: 15303.930 Da / Num. of mol.: 2 / Mutation: G103A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)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) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: P62799
#3: Protein Histone H2A type 1


Mass: 13978.241 Da / Num. of mol.: 2 / Mutation: G100R, A124S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: P06897
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13642.846 Da / Num. of mol.: 2 / Mutation: S33T, K121C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: P02281
#7: Protein Histone-lysine N-methyltransferase, H3 lysine-79 specific / Histone methyltransferase / DOT1-like protein / Histone H3-K79 methyltransferase / H3-K79-HMTase / Lysine N-methyltransferase 4


Mass: 47462.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOT1L, KIAA1814, KMT4 / Plasmid: pST50 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
References: UniProt: Q8TEK3, histone-lysine N-methyltransferase
#8: Protein Ubiquitin /


Mass: 8911.180 Da / Num. of mol.: 1 / Mutation: G76S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Plasmid: pST50 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: F5H388

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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 / Details: Widom 601 nucleosome positioning sequence / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (145-MER)


Mass: 44991.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Widom 601 nucleosome positioning sequence / 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

ComponentName: Dot1L-H2BK120ub nucleosome complex (Class 2A) / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.27 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.93 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy
Image recordingAverage exposure time: 60 sec. / Electron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1000

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.12model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 408526
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39558 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
11NW3

1nw3

A1NW311-332
21UBQ

1ubq

A1UBQ21-74
33LZ0

3lz0

A3LZ03
43LZ0

3lz0

B3LZ03
53LZ0

3lz0

C3LZ03
63LZ0

3lz0

D3LZ03
73LZ0

3lz0

E3LZ03
83LZ0

3lz0

F3LZ03
93LZ0

3lz0

G3LZ03
103LZ0

3lz0

H3LZ03
113LZ0

3lz0

I3LZ03
123LZ0

3lz0

J3LZ03

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