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- PDB-7xpx: Cryo-EM structure of the histone methyltransferase SET8 bound to ... -

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Basic information

Entry
Database: PDB / ID: 7xpx
TitleCryo-EM structure of the histone methyltransferase SET8 bound to H4K20Ecx-nucleosome
Components
  • (DNA (145-MER)) x 2
  • Histone H2A
  • Histone H2B 1.1
  • Histone H3
  • Histone H4
  • N-lysine methyltransferase KMT5A
KeywordsNUCLEAR PROTEIN/DNA / Methyltransferase / Nucleosome / NUCLEAR PROTEIN-DNA COMPLEX
Function / homology
Function and homology information


histone H4K20 monomethyltransferase activity / lysine N-methyltransferase activity / [histone H4]-lysine20 N-methyltransferase / histone H4K20 methyltransferase activity / histone H4 methyltransferase activity / peptidyl-lysine monomethylation / polytene chromosome / protein-lysine N-methyltransferase activity / mitotic chromosome condensation / regulation of DNA damage response, signal transduction by p53 class mediator ...histone H4K20 monomethyltransferase activity / lysine N-methyltransferase activity / [histone H4]-lysine20 N-methyltransferase / histone H4K20 methyltransferase activity / histone H4 methyltransferase activity / peptidyl-lysine monomethylation / polytene chromosome / protein-lysine N-methyltransferase activity / mitotic chromosome condensation / regulation of DNA damage response, signal transduction by p53 class mediator / histone methyltransferase activity / negative regulation of double-strand break repair via homologous recombination / Transferases; Transferring one-carbon groups; Methyltransferases / Condensation of Prophase Chromosomes / regulation of signal transduction by p53 class mediator / Regulation of TP53 Activity through Methylation / PKMTs methylate histone lysines / structural constituent of chromatin / transcription corepressor activity / nucleosome / protein heterodimerization activity / cell division / negative regulation of DNA-templated transcription / chromatin / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / DNA binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
Class V SAM-dependent methyltransferases / : / Histone-lysine N-methyltransferase (EC 2.1.1.43) family profile. / SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain / SET domain superfamily / SET domain / SET domain profile. / SET domain / Histone H2B signature. / Histone H2B ...Class V SAM-dependent methyltransferases / : / Histone-lysine N-methyltransferase (EC 2.1.1.43) family profile. / SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain / SET domain superfamily / SET domain / SET domain profile. / SET 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 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
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / DNA (> 100) / Histone H3 / Histone H2B 1.1 / Histone H2A / N-lysine methyltransferase KMT5A
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShi, L.X. / Zhou, Z.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: FASEB J / Year: 2022
Title: Structural basis of nucleosomal H4K20 methylation by methyltransferase SET8.
Authors: Liuxin Shi / Li Huang / Haizhen Long / Aoqun Song / Zheng Zhou /
Abstract: Histone H4 lysine 20 monomethylation (H4K20me1) plays a crucial role in multiple processes including DNA damage repair, DNA replication, and cell cycle control. Histone methyltransferase SET8 ...Histone H4 lysine 20 monomethylation (H4K20me1) plays a crucial role in multiple processes including DNA damage repair, DNA replication, and cell cycle control. Histone methyltransferase SET8 (previously named PR-Set7/KMT5A) mediates the chromatin deposition of H4K20me1, but how SET8 recognizes and modifies H4 in the context of the nucleosome is not fully understood. Here, we developed a simple chemical modification approach for H4K20 substitution by using the lysine analog S-ethyl-L-cysteine (Ecx). Substitution of H4K20 with H4Ecx20 improves the stability of the SET8-nucleosome complex, allowing us to determine the cryo-EM structure at 3.2 Å resolution. Structural analyses show that SET8 directly interacts with the H4 tail and the H2A-H2B acidic patch to ensure nucleosome binding. SET8 residues R339, K341, K351 make contact with nucleosomal DNA at the super helical location 2 (SHL2). Substitution of SET8 DNA-binding residues with alanines decreases the SET8-nucleosome interaction and impairs the methyltransferase activity. Disrupting the binding between SET8 R192 and H2A-H2B acidic patch decreases the cellular level of H4K20me1. Together, these results reveal a near-atomic resolution structure of SET8-bound nucleosome and provide insights into the SET8-mediated H4K20 recognition and modification. The lysine-to-Ecx substitution approach can be applied to the study of other methyltransferases.
History
DepositionMay 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3
B: Histone H4
C: Histone H2A
D: Histone H2B 1.1
E: Histone H3
F: Histone H4
G: Histone H2A
H: Histone H2B 1.1
I: DNA (145-MER)
J: DNA (145-MER)
K: N-lysine methyltransferase KMT5A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,40712
Polymers223,00811
Non-polymers3981
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 9 molecules AEBFCGDHK

#1: Protein Histone H3 /


Mass: 15303.930 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
#2: Protein Histone H4 /


Mass: 11265.247 Da / Num. of mol.: 2 / Fragment: K20(ECX)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli)
#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, XELAEV_18003602mg / Production host: Escherichia coli (E. coli) / References: UniProt: Q6AZJ8
#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) / References: UniProt: P02281
#7: Protein N-lysine methyltransferase KMT5A / H4-K20-HMTase KMT5A / Histone-lysine N-methyltransferase KMT5A / Lysine N-methyltransferase 5A / ...H4-K20-HMTase KMT5A / Histone-lysine N-methyltransferase KMT5A / Lysine N-methyltransferase 5A / Lysine-specific methylase 5A / PR/SET domain-containing protein 07 / PR-Set7 / PR/SET07 / SET domain-containing protein 8


Mass: 25349.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KMT5A, PRSET7, SET07, SET8, SETD8 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9NQR1, Transferases; Transferring one-carbon groups; Methyltransferases, [histone H4]-lysine20 N-methyltransferase

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-MER)


Mass: 44761.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#6: DNA chain DNA (145-MER)


Mass: 44752.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 1 types, 1 molecules

#8: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE / S-Adenosyl methionine


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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-IDSource
1Ternary complex of SET8 with H4K20Ecx nucleosome and SAMCOMPLEX#1-#70RECOMBINANT
2Histone H3/H4/H2A/H2BCOMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61RECOMBINANT
4N-lysine methyltransferase KMT5ACOMPLEX#71RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Xenopus laevis (African clawed frog)8355
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION / Nominal defocus max: 2200 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2227867
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 262534 / Symmetry type: POINT

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