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- PDB-8fyh: G4 RNA-mediated PRC2 dimer -

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Basic information

Entry
Database: PDB / ID: 8fyh
TitleG4 RNA-mediated PRC2 dimer
Components
  • (Polycomb protein ...Polycomb-group proteins) x 2
  • G4 RNA
  • Histone-binding protein RBBP4
  • Histone-lysine N-methyltransferase EZH2
  • Zinc finger protein AEBP2
  • protein Jumonji isoform X3
KeywordsGENE REGULATION / PRC2 / G-quadruplex RNA / RNP complex / chromatin modifier
Function / homology
Function and homology information


regulation of kidney development / hepatocyte homeostasis / cellular response to trichostatin A / regulation of gliogenesis / [histone H3]-lysine27 N-trimethyltransferase / negative regulation of striated muscle cell differentiation / sex chromatin / CAF-1 complex / negative regulation of keratinocyte differentiation / histone H3K27 trimethyltransferase activity ...regulation of kidney development / hepatocyte homeostasis / cellular response to trichostatin A / regulation of gliogenesis / [histone H3]-lysine27 N-trimethyltransferase / negative regulation of striated muscle cell differentiation / sex chromatin / CAF-1 complex / negative regulation of keratinocyte differentiation / histone H3K27 trimethyltransferase activity / negative regulation of retinoic acid receptor signaling pathway / random inactivation of X chromosome / primary miRNA binding / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / response to tetrachloromethane / cerebellar cortex development / histone H3K27 methyltransferase activity / facultative heterochromatin formation / positive regulation of cell cycle G1/S phase transition / NURF complex / regulation of cell fate specification / NuRD complex / negative regulation of stem cell population maintenance / DNA replication-dependent chromatin assembly / ESC/E(Z) complex / chromatin silencing complex / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / regulation of stem cell differentiation / protein-lysine N-methyltransferase activity / negative regulation of stem cell differentiation / Transcription of E2F targets under negative control by DREAM complex / RSC-type complex / pronucleus / cardiac muscle hypertrophy in response to stress / Polo-like kinase mediated events / synaptic transmission, GABAergic / G1 to G0 transition / : / lncRNA binding / positive regulation of dendrite development / histone H3 methyltransferase activity / negative regulation of gene expression, epigenetic / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / ATPase complex / positive regulation of stem cell population maintenance / Sin3-type complex / G1/S-Specific Transcription / histone methyltransferase activity / oligodendrocyte differentiation / negative regulation of transcription elongation by RNA polymerase II / Transcriptional Regulation by E2F6 / RNA Polymerase I Transcription Initiation / negative regulation of cell differentiation / subtelomeric heterochromatin formation / histone deacetylase complex / G0 and Early G1 / negative regulation of cytokine production involved in inflammatory response / RNA polymerase II core promoter sequence-specific DNA binding / pericentric heterochromatin / enzyme activator activity / heterochromatin formation / positive regulation of epithelial to mesenchymal transition / ribonucleoprotein complex binding / Cyclin E associated events during G1/S transition / Cyclin A:Cdk2-associated events at S phase entry / keratinocyte differentiation / Deposition of new CENPA-containing nucleosomes at the centromere / protein localization to chromatin / Regulation of TP53 Activity through Acetylation / methylated histone binding / SUMOylation of chromatin organization proteins / negative regulation of cell migration / B cell differentiation / transcription corepressor binding / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of PTEN gene transcription / Defective pyroptosis / HDACs deacetylate histones / liver regeneration / stem cell differentiation / promoter-specific chromatin binding / hippocampus development / transcription coregulator activity / negative regulation of transforming growth factor beta receptor signaling pathway / G1/S transition of mitotic cell cycle / brain development / positive regulation of MAP kinase activity / protein modification process / positive regulation of protein serine/threonine kinase activity / regulation of circadian rhythm / chromatin DNA binding / PKMTs methylate histone lysines / cellular response to hydrogen peroxide / positive regulation of GTPase activity / histone deacetylase binding / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / transcription corepressor activity
Similarity search - Function
EZH2, SET domain / Polycomb protein, VEFS-Box / VEFS-Box of polycomb protein / : / Ezh2, MCSS domain / Histone-lysine N-methyltransferase EZH1/EZH2 / Polycomb repressive complex 2 subunit EZH1/EZH2, tri-helical domain / Pre-SET CXC domain / WD repeat binding protein EZH2 / Polycomb repressive complex 2 tri-helical domain ...EZH2, SET domain / Polycomb protein, VEFS-Box / VEFS-Box of polycomb protein / : / Ezh2, MCSS domain / Histone-lysine N-methyltransferase EZH1/EZH2 / Polycomb repressive complex 2 subunit EZH1/EZH2, tri-helical domain / Pre-SET CXC domain / WD repeat binding protein EZH2 / Polycomb repressive complex 2 tri-helical domain / CXC domain / Tesmin/TSO1-like CXC domain / Tesmin/TSO1-like CXC domain / Histone-lysine N-methyltransferase EZH1/2-like / CXC domain / CXC domain profile. / Histone-binding protein RBBP4, N-terminal / Histone-binding protein RBBP4 or subunit C of CAF1 complex / Zinc finger, C5HC2-type / C5HC2 zinc finger / ARID/BRIGHT DNA binding domain / ARID DNA-binding domain / ARID DNA-binding domain superfamily / ARID/BRIGHT DNA binding domain / ARID domain profile. / BRIGHT, ARID (A/T-rich interaction domain) domain / JmjN domain / jmjN domain / JmjN domain profile. / Small domain found in the jumonji family of transcription factors / SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain / SET domain superfamily / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SET domain / SANT/Myb domain / SET domain profile. / SET domain / JmjC domain, hydroxylase / zinc finger / A domain family that is part of the cupin metalloenzyme superfamily. / JmjC domain / JmjC domain profile. / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 superfamily / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / Protein Jumonji isoform X3 / Polycomb protein EED / Histone-binding protein RBBP4 / Polycomb protein SUZ12 / Histone-lysine N-methyltransferase EZH2 / Zinc finger protein AEBP2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSong, J. / Kasinath, V.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM132544 United States
CitationJournal: Science / Year: 2023
Title: Structural basis for inactivation of PRC2 by G-quadruplex RNA.
Authors: Jiarui Song / Anne R Gooding / Wayne O Hemphill / Brittney D Love / Anne Robertson / Liqi Yao / Leonard I Zon / Trista E North / Vignesh Kasinath / Thomas R Cech /
Abstract: Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with ...Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme.
History
DepositionJan 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Polycomb protein SUZ12
C: Polycomb protein EED
D: Histone-binding protein RBBP4
A: Histone-lysine N-methyltransferase EZH2
E: protein Jumonji isoform X3
F: Zinc finger protein AEBP2
H: Polycomb protein SUZ12
I: Polycomb protein EED
J: Histone-binding protein RBBP4
G: Histone-lysine N-methyltransferase EZH2
K: protein Jumonji isoform X3
L: Zinc finger protein AEBP2
M: G4 RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)929,37127
Polymers928,45613
Non-polymers91614
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, MicroScale Thermophoresis (MST) was used to determine the stoichiometry of this complex
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Polycomb protein ... , 2 types, 4 molecules BHCI

#1: Protein Polycomb protein SUZ12 / Polycomb-group proteins / Chromatin precipitated E2F target 9 protein / ChET 9 protein / Joined to JAZF1 protein / Suppressor ...Chromatin precipitated E2F target 9 protein / ChET 9 protein / Joined to JAZF1 protein / Suppressor of zeste 12 protein homolog


Mass: 83181.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SUZ12, CHET9, JJAZ1, KIAA0160 / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15022
#2: Protein Polycomb protein EED / / hEED / Embryonic ectoderm development protein / WD protein associating with integrin cytoplasmic ...hEED / Embryonic ectoderm development protein / WD protein associating with integrin cytoplasmic tails 1 / WAIT-1


Mass: 50267.691 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EED / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75530

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Protein , 4 types, 8 molecules DJAGEKFL

#3: Protein Histone-binding protein RBBP4 / Chromatin assembly factor 1 subunit C / CAF-1 subunit C / Chromatin assembly factor I p48 subunit / ...Chromatin assembly factor 1 subunit C / CAF-1 subunit C / Chromatin assembly factor I p48 subunit / CAF-I p48 / Nucleosome-remodeling factor subunit RBAP48 / Retinoblastoma-binding protein 4 / RBBP-4 / Retinoblastoma-binding protein p48


Mass: 47709.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBBP4, RBAP48 / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q09028
#4: Protein Histone-lysine N-methyltransferase EZH2 / ENX-1 / Enhancer of zeste homolog 2 / Lysine N-methyltransferase 6


Mass: 86149.055 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EZH2, KMT6 / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q15910, [histone H3]-lysine27 N-trimethyltransferase
#5: Protein protein Jumonji isoform X3


Mass: 138406.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: Jarid2 / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A6I9KXB3
#6: Protein Zinc finger protein AEBP2 / / Adipocyte enhancer-binding protein 2 / AE-binding protein 2


Mass: 54535.496 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AEBP2 / Cell line (production host): BTI-TN5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q6ZN18

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RNA chain / Non-polymers , 2 types, 15 molecules M

#7: RNA chain G4 RNA


Mass: 7955.823 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: G4 RNA-mediated dimer of polycomb repressive complex 2
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.8 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: BTI-TN5B1-4
Buffer solutionpH: 7.9
Details: RNP complex buffer (25 mM HEPES pH 7.9, 50 mM KCl, 2 mM MgCl2, 10% glycerol, and 1mM TCEP) EM preparation buffer I (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, and 1mM TCEP) EM preparation ...Details: RNP complex buffer (25 mM HEPES pH 7.9, 50 mM KCl, 2 mM MgCl2, 10% glycerol, and 1mM TCEP) EM preparation buffer I (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, and 1mM TCEP) EM preparation buffer II (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, 0.01%NP-40, and 1mM TCEP).
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
250 mMPotassium chlorideKCl1
32.5 %GlycerolC3H8O31
40.01 %np-40 substitute1
51 mMTCEPC9H15O6P1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: We used streptavidin-affinity grid preparation method with biotin-labeled RNA at 100 nM concentration. PRC2 was applied in excess at 600 nM.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 281 K / Details: 3s of single side blotting

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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: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18632 / Details: 60 frames per movie
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1RELION4.0-beta-2particle selection
2SerialEMimage acquisition
4RELION4.0-beta-2CTF correction
7Cootmodel fitting
9RELION4.0-beta-2initial Euler assignment
10RELION4.0-beta-2final Euler assignment
11RELION4.0-beta-2classification
12RELION4.0-beta-23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3885383
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217196 / Symmetry type: POINT

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