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- PDB-6o96: Dot1L bound to the H2BK120 Ubiquitinated nucleosome -

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Basic information

Entry
Database: PDB / ID: 6o96
TitleDot1L bound to the H2BK120 Ubiquitinated nucleosome
Components
  • (DNA (146-MER)) x 2
  • Histone H2A
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
  • Histone-lysine N-methyltransferase, H3 lysine-79 specific
  • Polyubiquitin-B
KeywordsStructural Protein/DNA/Transferase / Complex / Chromatin Modifier / Structural Protein / TRANSFERASE / Structural Protein-DNA-Transferase complex
Function / homology
Function and homology information


[histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / histone H3K79 trimethyltransferase activity / regulation of transcription regulatory region DNA binding / symbiont entry into host cell via disruption of host cell glycocalyx / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / symbiont entry into host cell via disruption of host cell envelope / histone methyltransferase activity / virus tail ...[histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / histone H3K79 trimethyltransferase activity / regulation of transcription regulatory region DNA binding / symbiont entry into host cell via disruption of host cell glycocalyx / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / symbiont entry into host cell via disruption of host cell envelope / histone methyltransferase activity / virus tail / subtelomeric heterochromatin formation / telomere organization / DNA damage checkpoint signaling / PKMTs methylate histone lysines / structural constituent of chromatin / nucleosome / heterochromatin formation / nucleosome assembly / methylation / gene expression / nucleic acid binding / RNA polymerase II-specific DNA-binding transcription factor binding / transcription coactivator activity / chromosome, telomeric region / protein heterodimerization activity / DNA repair / intracellular membrane-bounded organelle / 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. / 434 Repressor (Amino-terminal Domain) / Histone, subunit A / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like ...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. / 434 Repressor (Amino-terminal Domain) / Histone, subunit A / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Histone, subunit A / Pectin lyase fold / Pectin lyase fold/virulence factor / Vaccinia Virus protein VP39 / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / : / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin conserved site / 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 / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / 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
S-ADENOSYL-L-HOMOCYSTEINE / DNA / DNA (> 10) / DNA (> 100) / Ubiquitin B / Histone H2B 1.1 / Histone H2A type 1 / Tail fiber / Histone H4 / Histone H3.2 ...S-ADENOSYL-L-HOMOCYSTEINE / DNA / DNA (> 10) / DNA (> 100) / Ubiquitin B / Histone H2B 1.1 / Histone H2A type 1 / Tail fiber / Histone H4 / Histone H3.2 / Histone H2A / Histone-lysine N-methyltransferase, H3 lysine-79 specific
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
synthetic construct (others)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsValencia-Sanchez, M.I. / De Ioannes, P.E. / Miao, W. / Vasilyev, N. / Chen, R. / Nudler, E. / Armache, J.-P. / Armache, K.-J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM115882-03 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127267 United States
Other privateDavid and Lucile Packard Foundation United States
CitationJournal: Mol Cell / Year: 2019
Title: Structural Basis of Dot1L Stimulation by Histone H2B Lysine 120 Ubiquitination.
Authors: Marco Igor Valencia-Sánchez / Pablo De Ioannes / Miao Wang / Nikita Vasilyev / Ruoyu Chen / Evgeny Nudler / Jean-Paul Armache / Karim-Jean Armache /
Abstract: The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of ...The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.
History
DepositionMar 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization
Revision 1.3Feb 1, 2023Group: Database references / Refinement description
Category: citation_author / database_2 / pdbx_initial_refinement_model
Item: _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4May 15, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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
Revision 1.5Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp / em_entity_assembly_molwt / pdbx_entry_details
Item: _chem_comp.mon_nstd_flag / _chem_comp.type / _em_entity_assembly_molwt.entity_assembly_id

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Structure visualization

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  • Deposited structure unit
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  • EMDB-0652
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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 (146-MER)
J: DNA (146-MER)
K: Histone-lysine N-methyltransferase, H3 lysine-79 specific
L: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)246,25813
Polymers245,87312
Non-polymers3841
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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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: G102A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P84233
#2: Protein Histone H4


Mass: 11394.426 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
#3: Protein Histone H2A


Mass: 14109.436 Da / Num. of mol.: 2 / Mutation: G99R, A123S
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 1.1 / H2B1.1


Mass: 13629.911 Da / Num. of mol.: 2 / Mutation: K120C, S32T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281
#7: Protein Histone-lysine N-methyltransferase, H3 lysine-79 specific / DOT1-like protein / Histone H3-K79 methyltransferase / H3-K79-HMTase / Lysine N-methyltransferase 4


Mass: 38245.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOT1L, KIAA1814, KMT4 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8TEK3, histone-lysine N-methyltransferase
#8: Protein Polyubiquitin-B


Mass: 8622.922 Da / Num. of mol.: 1 / Mutation: G76C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: J3QS39, UniProt: P0CG47*PLUS

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (146-MER)


Mass: 44824.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain DNA (146-MER)


Mass: 45304.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 1 types, 1 molecules

#9: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S

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Details

Has protein modificationN

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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
IDNameTypeEntity IDParent-IDSourceDetails
13.5 angstroms Dot1L-H2BK120Ub Sample 1ACOMPLEX#1-#80RECOMBINANT
2Ubiquitinated NucleosomeCOMPLEX#1-#6, #81RECOMBINANTUbiquitinated Nucleosome
3Histone-lysine N-methyltransferase H3 lysine-79 specificCOMPLEX#71RECOMBINANTHistone-lysine N-methyltransferase H3 lysine-79 specific
4UbiquitinCOMPLEX#82RECOMBINANTUbiquitin
5Histone octamerCOMPLEX#1-#42RECOMBINANTHistone octamer
6DNA 146-MERCOMPLEX#5-#62RECOMBINANTDNA 146-MER
Molecular weight
IDEntity assembly-IDUnitsExperimental value
11MEGADALTONSNO
22NO
33NO
14
15
16
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Xenopus laevis (African clawed frog)8355
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Xenopus laevis (African clawed frog)8355
76synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
65Escherichia coli (E. coli)562
76Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3.5 angstroms Dot1L-H2BK120Ub Sample 1A
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 297.15 K / Details: blotted for 3s before plunging

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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: 3200 nm / Nominal defocus min: 1700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1.1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2Scipionimage acquisition
4Gctfv1CTF correction
7UCSF Chimera1.13.1model fitting
11RELIONclassification
12cisTEM3D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 211279 / Symmetry type: POINT
Atomic model buildingB value: 91.61 / Protocol: OTHER / Space: REAL
Atomic model building

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

IDPDB-IDAccession codeInitial refinement model-ID
11NW3

1nw3

1NW31
21UBQ

1ubq

1UBQ2
33TU4

3tu4

3TU43
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01116091
ELECTRON MICROSCOPYf_angle_d0.96623022
ELECTRON MICROSCOPYf_dihedral_angle_d21.5978661
ELECTRON MICROSCOPYf_chiral_restr0.0562618
ELECTRON MICROSCOPYf_plane_restr0.0081914

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