+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29578 | |||||||||
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Title | G4 RNA-mediated PRC2 dimer | |||||||||
Map data | consensus map of G4 RNA-mediated PRC2 dimer | |||||||||
Sample |
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Keywords | PRC2 / G-quadruplex RNA / RNP complex / chromatin modifier / GENE REGULATION | |||||||||
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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Song J / Kasinath V | |||||||||
Funding support | United States, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29578.map.gz | 80.2 MB | EMDB map data format | |
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Header (meta data) | emd-29578-v30.xml emd-29578.xml | 31.6 KB 31.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29578_fsc.xml emd_29578_fsc_2.xml emd_29578_fsc_3.xml | 10.7 KB 10.7 KB 10.6 KB | Display Display Display | FSC data file |
Images | emd_29578.png | 117.5 KB | ||
Masks | emd_29578_msk_1.map | 103 MB | Mask map | |
Filedesc metadata | emd-29578.cif.gz | 9.7 KB | ||
Others | emd_29578_additional_1.map.gz emd_29578_half_map_1.map.gz emd_29578_half_map_2.map.gz | 8.3 MB 80.9 MB 80.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29578 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29578 | HTTPS FTP |
-Related structure data
Related structure data | 8fyhMC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29578.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | consensus map of G4 RNA-mediated PRC2 dimer | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.06344 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_29578_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: consensus map of G4 RNA-mediated PRC2 dimer after...
File | emd_29578_additional_1.map | ||||||||||||
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Annotation | consensus map of G4 RNA-mediated PRC2 dimer after postprocessing with B-factor of -40 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 2 of consensus map
File | emd_29578_half_map_1.map | ||||||||||||
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Annotation | half map 2 of consensus map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 1 of consensus map
File | emd_29578_half_map_2.map | ||||||||||||
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Annotation | half map 1 of consensus map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : G4 RNA-mediated dimer of polycomb repressive complex 2
Entire | Name: G4 RNA-mediated dimer of polycomb repressive complex 2 |
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Components |
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-Supramolecule #1: G4 RNA-mediated dimer of polycomb repressive complex 2
Supramolecule | Name: G4 RNA-mediated dimer of polycomb repressive complex 2 type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 800 KDa |
-Macromolecule #1: Polycomb protein SUZ12
Macromolecule | Name: Polycomb protein SUZ12 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 83.181922 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MAPQKHGGGG GGGSGPSAGS GGGGFGGSAA VAAATASGGK SGGGSCGGGG SYSASSSSSA AAAAGAAVLP VKKPKMEHVQ ADHELFLQA FEKPTQIYRF LRTRNLIAPI FLHRTLTYMS HRNSRTNIKR KTFKVDDMLS KVEKMKGEQE SHSLSAHLQL T FTGFFHKN ...String: MAPQKHGGGG GGGSGPSAGS GGGGFGGSAA VAAATASGGK SGGGSCGGGG SYSASSSSSA AAAAGAAVLP VKKPKMEHVQ ADHELFLQA FEKPTQIYRF LRTRNLIAPI FLHRTLTYMS HRNSRTNIKR KTFKVDDMLS KVEKMKGEQE SHSLSAHLQL T FTGFFHKN DKPSPNSENE QNSVTLEVLL VKVCHKKRKD VSCPIRQVPT GKKQVPLNPD LNQTKPGNFP SLAVSSNEFE PS NSHMVKS YSLLFRVTRP GRREFNGMIN GETNENIDVN EELPARRKRN REDGEKTFVA QMTVFDKNRR LQLLDGEYEV AMQ EMEECP ISKKRATWET ILDGKRLPPF ETFSQGPTLQ FTLRWTGETN DKSTAPIAKP LATRNSESLH QENKPGSVKP TQTI AVKES LTTDLQTRKE KDTPNENRQK LRIFYQFLYN NNTRQQTEAR DDLHCPWCTL NCRKLYSLLK HLKLCHSRFI FNYVY HPKG ARIDVSINEC YDGSYAGNPQ DIHRQPGFAF SRNGPVKRTP ITHILVCRPK RTKASMSEFL ESEDGEVEQQ RTYSSG HNR LYFHSDTCLP LRPQEMEVDS EDEKDPEWLR EKTITQIEEF SDVNEGEKEV MKLWNLHVMK HGFIADNQMN HACMLFV EN YGQKIIKKNL CRNFMLHLVS MHDFNLISIM SIDKAVTKLR EMQQKLEKGE SASPANEEIT EEQNGTANGF SEINSKEK A LETDSVSGVS KQSKKQKL UniProtKB: Polycomb protein SUZ12 |
-Macromolecule #2: Polycomb protein EED
Macromolecule | Name: Polycomb protein EED / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 50.267691 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MSEREVSTAP AGTDMPAAKK QKLSSDENSN PDLSGDENDD AVSIESGTNT ERPDTPTNTP NAPGRKSWGK GKWKSKKCKY SFKCVNSLK EDHNQPLFGV QFNWHSKEGD PLVFATVGSN RVTLYECHSQ GEIRLLQSYV DADADENFYT CAWTYDSNTS H PLLAVAGS ...String: MSEREVSTAP AGTDMPAAKK QKLSSDENSN PDLSGDENDD AVSIESGTNT ERPDTPTNTP NAPGRKSWGK GKWKSKKCKY SFKCVNSLK EDHNQPLFGV QFNWHSKEGD PLVFATVGSN RVTLYECHSQ GEIRLLQSYV DADADENFYT CAWTYDSNTS H PLLAVAGS RGIIRIINPI TMQCIKHYVG HGNAINELKF HPRDPNLLLS VSKDHALRLW NIQTDTLVAI FGGVEGHRDE VL SADYDLL GEKIMSCGMD HSLKLWRINS KRMMNAIKES YDYNPNKTNR PFISQKIHFP DFSTRDIHRN YVDCVRWLGD LIL SKSCEN AIVCWKPGKM EDDIDKIKPS ESNVTILGRF DYSQCDIWYM RFSMDFWQKM LALGNQVGKL YVWDLEVEDP HKAK CTTLT HHKCGAAIRQ TSFSRDSSIL IAVCDDASIW RWDRLR UniProtKB: Polycomb protein EED |
-Macromolecule #3: Histone-binding protein RBBP4
Macromolecule | Name: Histone-binding protein RBBP4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 47.709527 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MADKEAAFDD AVEERVINEE YKIWKKNTPF LYDLVMTHAL EWPSLTAQWL PDVTRPEGKD FSIHRLVLGT HTSDEQNHLV IASVQLPND DAQFDASHYD SEKGEFGGFG SVSGKIEIEI KINHEGEVNR ARYMPQNPCI IATKTPSSDV LVFDYTKHPS K PDPSGECN ...String: MADKEAAFDD AVEERVINEE YKIWKKNTPF LYDLVMTHAL EWPSLTAQWL PDVTRPEGKD FSIHRLVLGT HTSDEQNHLV IASVQLPND DAQFDASHYD SEKGEFGGFG SVSGKIEIEI KINHEGEVNR ARYMPQNPCI IATKTPSSDV LVFDYTKHPS K PDPSGECN PDLRLRGHQK EGYGLSWNPN LSGHLLSASD DHTICLWDIS AVPKEGKVVD AKTIFTGHTA VVEDVSWHLL HE SLFGSVA DDQKLMIWDT RSNNTSKPSH SVDAHTAEVN CLSFNPYSEF ILATGSADKT VALWDLRNLK LKLHSFESHK DEI FQVQWS PHNETILASS GTDRRLNVWD LSKIGEEQSP EDAEDGPPEL LFIHGGHTAK ISDFSWNPNE PWVICSVSED NIMQ VWQMA ENIYNDEDPE GSVDPEGQGS UniProtKB: Histone-binding protein RBBP4 |
-Macromolecule #4: Histone-lysine N-methyltransferase EZH2
Macromolecule | Name: Histone-lysine N-methyltransferase EZH2 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: [histone H3]-lysine27 N-trimethyltransferase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 86.149055 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MGQTGKKSEK GPVCWRKRVK SEYMRLRQLK RFRRADEVKS MFSSNRQKIL ERTEILNQEW KQRRIQPVHI LTSVSSLRGT RECSVTSDL DFPTQVIPLK TLNAVASVPI MYSWSPLQQN FMVEDETVLH NIPYMGDEVL DQDGTFIEEL IKNYDGKVHG D RECGFIND ...String: MGQTGKKSEK GPVCWRKRVK SEYMRLRQLK RFRRADEVKS MFSSNRQKIL ERTEILNQEW KQRRIQPVHI LTSVSSLRGT RECSVTSDL DFPTQVIPLK TLNAVASVPI MYSWSPLQQN FMVEDETVLH NIPYMGDEVL DQDGTFIEEL IKNYDGKVHG D RECGFIND EIFVELVNAL GQYNDDDDDD DGDDPEEREE KQKDLEDHRD DKESRPPRKF PSDKIFEAIS SMFPDKGTAE EL KEKYKEL TEQQLPGALP PECTPNIDGP NAKSVQREQS LHSFHTLFCR RCFKYDCFLH RKCNYSFHAT PNTYKRKNTE TAL DNKPCG PQCYQHLEGA KEFAAALTAE RIKTPPKRPG GRRRGRLPNN SSRPSTPTIN VLESKDTDSD REAGTETGGE NNDK EEEEK KDETSSSSEA NSRCQTPIKM KPNIEPPENV EWSGAEASMF RVLIGTYYDN FCAIARLIGT KTCRQVYEFR VKESS IIAP APAEDVDTPP RKKKRKHRLW AAHCRKIQLK KDGSSNHVYN YQPCDHPRQP CDSSCPCVIA QNFCEKFCQC SSECQN RFP GCRCKAQCNT KQCPCYLAVR ECDPDLCLTC GAADHWDSKN VSCKNCSIQR GSKKHLLLAP SDVAGWGIFI KDPVQKN EF ISEYCGEIIS QDEADRRGKV YDKYMCSFLF NLNNDFVVDA TRKGNKIRFA NHSVNPNCYA KVMMVNGDHR IGIFAKRA I QTGEELFFDY RYSQADALKY VGIEREMEIP UniProtKB: Histone-lysine N-methyltransferase EZH2 |
-Macromolecule #5: protein Jumonji isoform X3
Macromolecule | Name: protein Jumonji isoform X3 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 138.406219 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MSKERPKRNI IQKKYDDSDG IPWSEERVVR KVLYLSLKEF KNAQKRQHGE GIAGSLKSVN GLLGNDQSKA LGPASEQSEN EKDDASQVS STSNDVSSSD FEEGPSRKRP RLQAQRKFAQ SQPNSPSTTP VKTVEPLLPP PATQISDLSK RKPKTEDFLT F LCLRGSPA ...String: MSKERPKRNI IQKKYDDSDG IPWSEERVVR KVLYLSLKEF KNAQKRQHGE GIAGSLKSVN GLLGNDQSKA LGPASEQSEN EKDDASQVS STSNDVSSSD FEEGPSRKRP RLQAQRKFAQ SQPNSPSTTP VKTVEPLLPP PATQISDLSK RKPKTEDFLT F LCLRGSPA LPSSMVYFGS SQDEEDVEEE DDETEDVKTA NNNASSSCQS TPRKGKTHKH VHNGHVFNGS NRSTREKEPA QK HKSKETT PAKEKHIDHR ADSRREPASV AQPTATPSAG SLAKGLPANH QPPPPHRSAQ DLRKQVTLHV SKVNGVTRMS SLG AGTTSA KKIREVRPSP SKTVKYTATV TKGTVTYTKA KRELVKETKP THHKPSSAVN HTISGKTESS NAKTRKQVLS LGGA STSTG PAASGLKASS RLNPKSCTKE VGGRQLREGL RNSKRRLEEA QQVDKPQSPP KKMKGAAGIA EAPGKKASAA SAEKS LLNG HVKKEVPERS LERNRPKRAT AGKNMPGKQA HGKAEGTPCE NRSTSQPESS HKPHDPQGKP EKGIGKSGWT AMDEIP VLR PSAKEFHDPL IYIESVRAQV EKYGMCRVIP PPDWRPECKL NDEMRFVTQI QHIHKLGRRW GPNVQRLACI KKHLRSQ GI TMDELPLIGG CELDLACFFR LINEMGGMQQ VTDLKKWNKL ADMLRIPKTA QDRLAKLQEA YCQYLLSYDS LSPEEHRR L EKEVLMEKEI LEKRKGPLEG HTENDHHKFH SLPRFEPKNG LIHGVTPRNG FRSKLKEVGQ APLKTGRRRL FAQEKEVVK EEEEDKGVLN DFHKCIYKGR SVSLTTFYRT ARNIMNMCFS KEPAPAEIEQ EYWRLVEEKD CHVAVHCGKV DTNTHGSGFP VGKSEPFSR HGWNLTVLPN NTGSILRHLG AVPGVTIPWL NIGMVFSTSC WSRDQNHLPY IDYLHTGADC IWYCIPAEEE N KLEDVVHT LLQANGTPGL QMLESNVMIS PEVLCKEGIK VHRTVQQSGQ FVVCFPGSFV SKVCCGYSVS ETVHFATTQW TS MGFETAK EMKRRHIAKP FSMEKLLYQI AQAEAKKENG PTLSTISALL DELRDTELRQ RRQLFEAGLH SSARYGSHDG NST VADGKK KPRKWLQLET SERRCQICQH LCYLSMVVQE NENVVFCLEC ALRHVEKQKS CRGLKLMYRY DEEQIISLVN QICG KVSGK HGGIENCLNK PTPKRGPRKR ATVDVPPSRL PSS UniProtKB: Protein Jumonji isoform X3 |
-Macromolecule #6: Zinc finger protein AEBP2
Macromolecule | Name: Zinc finger protein AEBP2 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 54.535496 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MAAAITDMAD LEELSRLSPL PPGSPGSAAR GRAEPPEEEE EEEEEEEEAE AEAVAALLLN GGSGGGGGGG GGGVGGGEAE TMSEPSPES ASQAGEDEDE EEDDEEEEDE SSSSGGGEEE SSAESLVGSS GGSSSDETRS LSPGAASSSS GDGDGKEGLE E PKGPRGSQ ...String: MAAAITDMAD LEELSRLSPL PPGSPGSAAR GRAEPPEEEE EEEEEEEEAE AEAVAALLLN GGSGGGGGGG GGGVGGGEAE TMSEPSPES ASQAGEDEDE EEDDEEEEDE SSSSGGGEEE SSAESLVGSS GGSSSDETRS LSPGAASSSS GDGDGKEGLE E PKGPRGSQ GGGGGGSSSS SVVSSGGDEG YGTGGGGSSA TSGGRRGSLE MSSDGEPLSR MDSEDSISST IMDVDSTISS GR STPAMMN GQGSTTSSSK NIAYNCCWDQ CQACFNSSPD LADHIRSIHV DGQRGGVFVC LWKGCKVYNT PSTSQSWLQR HML THSGDK PFKCVVGGCN ASFASQGGLA RHVPTHFSQQ NSSKVSSQPK AKEESPSKAG MNKRRKLKNK RRRSLPRPHD FFDA QTLDA IRHRAICFNL SAHIESLGKG HSVVFHSTVI AKRKEDSGKI KLLLHWMPED ILPDVWVNES ERHQLKTKVV HLSKL PKDT ALLLDPNIYR TMPQKRLKRT LIRKVFNLYL SKQ UniProtKB: Zinc finger protein AEBP2 |
-Macromolecule #7: G4 RNA
Macromolecule | Name: G4 RNA / type: rna / ID: 7 / Number of copies: 1 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 7.955823 KDa |
Sequence | String: GGGUAAGGGU AAGGGUAAGG GUAA |
-Macromolecule #8: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 14 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.9 Component:
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). | ||||||||||||||||||
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Grid | Model: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 1.5 | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 281 K / Instrument: LEICA PLUNGER / Details: 3s of single side blotting. | ||||||||||||||||||
Details | We used streptavidin-affinity grid preparation method with biotin-labeled RNA at 100 nM concentration. PRC2 was applied in excess at 600 nM. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000 |
Specialist optics | Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 18632 / Average electron dose: 60.0 e/Å2 / Details: 60 frames per movie |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |