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- EMDB-20553: Cryo-EM structure of DDB1dB-DCAF15-DDA1 bound to E7820 and RBM39 -

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Basic information

Entry
Database: EMDB / ID: EMD-20553
TitleCryo-EM structure of DDB1dB-DCAF15-DDA1 bound to E7820 and RBM39
Map dataFinal refinement map
Sample
  • Complex: Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: DDB1 and CUL4 associated factor 15
    • Protein or peptide: RNA binding motif protein 39
    • Protein or peptide: DET1 And DDB1 Associated 1
Function / homology
Function and homology information


RS domain binding / regulation of natural killer cell activation / positive regulation by virus of viral protein levels in host cell / U1 snRNP binding / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / regulation of mRNA splicing, via spliceosome / biological process involved in interaction with symbiont ...RS domain binding / regulation of natural killer cell activation / positive regulation by virus of viral protein levels in host cell / U1 snRNP binding / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / regulation of mRNA splicing, via spliceosome / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / immune system process / negative regulation of reproductive process / negative regulation of developmental process / cullin family protein binding / small molecule binding / centriolar satellite / viral release from host cell / ectopic germ cell programmed cell death / positive regulation of viral genome replication / RNA processing / positive regulation of gluconeogenesis / mRNA Splicing - Major Pathway / RNA splicing / proteasomal protein catabolic process / Recognition of DNA damage by PCNA-containing replication complex / nucleotide-excision repair / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / regulation of circadian rhythm / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / mRNA processing / Wnt signaling pathway / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / protein polyubiquitination / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / microtubule cytoskeleton / protein-macromolecule adaptor activity / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / site of double-strand break / Neddylation / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / chromosome, telomeric region / protein ubiquitination / nuclear speck / DNA repair / apoptotic process / DNA damage response / protein-containing complex binding / nucleolus / negative regulation of apoptotic process / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
DDB1- and CUL4-associated factor 15, WD40 repeat-containing domain / DDB1- and CUL4-associated factor 15 / : / DDB1-and CUL4-substrate receptor 15, WD repeat / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / Splicing factor, RBM39-like / Splicing factor RBM39, linker / linker between RRM2 and RRM3 domains in RBM39 protein ...DDB1- and CUL4-associated factor 15, WD40 repeat-containing domain / DDB1- and CUL4-associated factor 15 / : / DDB1-and CUL4-substrate receptor 15, WD repeat / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / Splicing factor, RBM39-like / Splicing factor RBM39, linker / linker between RRM2 and RRM3 domains in RBM39 protein / RNA recognition motif domain, eukaryote / RNA recognition motif / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
RNA-binding protein 39 / DNA damage-binding protein 1 / DDB1- and CUL4-associated factor 15 / DET1- and DDB1-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsFaust T / Yoon H / Nowak RP / Donovan KA / Li Z / Cai Q / Eleuteri NA / Zhang T / Gray NS / Fischer ES
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteR01CA214608 United States
CitationJournal: Nat Chem Biol / Year: 2020
Title: Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15.
Authors: Tyler B Faust / Hojong Yoon / Radosław P Nowak / Katherine A Donovan / Zhengnian Li / Quan Cai / Nicholas A Eleuteri / Tinghu Zhang / Nathanael S Gray / Eric S Fischer /
Abstract: The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically ...The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 Å, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.
History
DepositionAug 2, 2019-
Header (metadata) releaseAug 14, 2019-
Map releaseNov 20, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20553.png

Downloads & links

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Map

FileDownload / File: emd_20553.map.gz / Format: CCP4 / Size: 50.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal refinement map
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 0.0119 / Movie #1: 0.0119
Minimum - Maximum-0.033653937 - 0.04070471
Average (Standard dev.)0.00002029801 (±0.0015149929)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions236236236
Spacing236236236
CellA=B=C: 249.92401 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z236236236
origin x/y/z0.0000.0000.000
length x/y/z249.924249.924249.924
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ172172172
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS236236236
D min/max/mean-0.0340.0410.000

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Supplemental data

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Mask #1

Fileemd_20553_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Sharpened refinement map

Fileemd_20553_additional.map
AnnotationSharpened refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened refinement map

Fileemd_20553_additional_1.map
AnnotationSharpened refinement map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #1

Fileemd_20553_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_20553_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39

EntireName: Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39
Components
  • Complex: Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: DDB1 and CUL4 associated factor 15
    • Protein or peptide: RNA binding motif protein 39
    • Protein or peptide: DET1 And DDB1 Associated 1

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Supramolecule #1: Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39

SupramoleculeName: Complex of DDB1-DCAF15-DDA1 bound to E7820 and the second RRM of RBM39
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Compound: E7820; Chemical Name: 3-Cyano-N-(3-cyano-4-methyl-1H-indol-7-yl)benzenesulfonamide
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 193 KDa

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Macromolecule #1: DNA damage-binding protein 1

MacromoleculeName: DNA damage-binding protein 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSSHHHHHH SAAHIVMVDA YKPTKGGRMS YNYVVTAQKP TAVNGCVTGH FTSAEDLNLL IAKNTRLEIY VVTAEGLRPV KEVGMYGKIA VMELFRPKGE SKDLLFILTA KYNACILEYK QSGESIDIIT RAHGNVQDRI GRPSETGIIG IIDPECRMIG LRLYDGLFKV ...String:
MGSSHHHHHH SAAHIVMVDA YKPTKGGRMS YNYVVTAQKP TAVNGCVTGH FTSAEDLNLL IAKNTRLEIY VVTAEGLRPV KEVGMYGKIA VMELFRPKGE SKDLLFILTA KYNACILEYK QSGESIDIIT RAHGNVQDRI GRPSETGIIG IIDPECRMIG LRLYDGLFKV IPLDRDNKEL KAFNIRLEEL HVIDVKFLYG CQAPTICFVY QDPQGRHVKT YEVSLREKEF NKGPWKQENV EAEASMVIAV PEPFGGAIII GQESITYHNG DKYLAIAPPI IKQSTIVCHN RVDPNGSRYL LGDMEGRLFM LLLEKEEQMD GTVTLKDLRV ELLGETSIAE CLTYLDNGVV FVGSRLGDSQ LVKLNVDSNE QGSYVVAMET FTNLGPIVDM CVVDLERQGQ GQLVTCSGAF KEGSLRIIRN GIGGNGNSGE IQKLHIRTVP LYESPRKICY QEVSQCFGVL SSRIEVQDTS GGTTALRPSA STQALSSSVS SSKLFSSSTA PHETSFGEEV EVHNLLIIDQ HTFEVLHAHQ FLQNEYALSL VSCKLGKDPN TYFIVGTAMV YPEEAEPKQG RIVVFQYSDG KLQTVAEKEV KGAVYSMVEF NGKLLASINS TVRLYEWTTE KELRTECNHY NNIMALYLKT KGDFILVGDL MRSVLLLAYK PMEGNFEEIA RDFNPNWMSA VEILDDDNFL GAENAFNLFV CQKDSAATTD EERQHLQEVG LFHLGEFVNV FCHGSLVMQN LGETSTPTQG SVLFGTVNGM IGLVTSLSES WYNLLLDMQN RLNKVIKSVG KIEHSFWRSF HTERKTEPAT GFIDGDLIES FLDISRPKMQ EVVANLQYDD GSGMKREATA DDLIKVVEEL TRIH

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Macromolecule #2: DDB1 and CUL4 associated factor 15

MacromoleculeName: DDB1 and CUL4 associated factor 15 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDWSHPQFEK SAVDENLYFQ GGGRMAPSSK SERNSGAGSG GGGPGGAGGK RAAGRRREHV LKQLERVKIS GQLSPRLFRK LPPRVCVSLK NIVDEDFLYA GHIFLGFSKC GRYVLSYTSS SGDDDFSFYI YHLYWWEFNV HSKLKLVRQV RLFQDEEIYS DLYLTVCEWP ...String:
MDWSHPQFEK SAVDENLYFQ GGGRMAPSSK SERNSGAGSG GGGPGGAGGK RAAGRRREHV LKQLERVKIS GQLSPRLFRK LPPRVCVSLK NIVDEDFLYA GHIFLGFSKC GRYVLSYTSS SGDDDFSFYI YHLYWWEFNV HSKLKLVRQV RLFQDEEIYS DLYLTVCEWP SDASKVIVFG FNTRSANGML MNMMMMSDEN HRDIYVSTVA VPPPGRCAAC QDASRAHPGD PNAQCLRHGF MLHTKYQVVY PFPTFQPAFQ LKKDQVVLLN TSYSLVACAV SVHSAGDRSF CQILYDHSTC PLAPASPPEP QSPELPPALP SFCPEAAPAR SSGSPEPSPA IAKAKEFVAD IFRRAKEAKG GVPEEARPAL CPGPSGSRCR AHSEPLALCG ETAPRDSPPA SEAPASEPGY VNYTKLYYVL ESGEGTEPED ELEDDKISLP FVVTDLRGRN LRPMRERTAV QGQYLTVEQL TLDFEYVINE VIRHDATWGH QFCSFSDYDI VILEVCPETN QVLINIGLLL LAFPSPTEEG QLRPKTYHTS LKVAWDLNTG IFETVSVGDL TEVKGQTSGS VWSSYRKSCV DMVMKWLVPE SSGRYVNRMT NEALHKGCSL KVLADSERYT WIVL

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Macromolecule #4: RNA binding motif protein 39

MacromoleculeName: RNA binding motif protein 39 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MGSSHHHHHH SAVDENLYFQ GGCGRGSAGP MRLYVGSLHF NITEDMLRGI FEPFGRIESI QLMMDSETGR SKGYGFITFS DSECAKKALE QLNGFELAGR PMKVGHVTER TDA

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Macromolecule #5: DET1 And DDB1 Associated 1

MacromoleculeName: DET1 And DDB1 Associated 1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MGSSHHHHHH SAVDENLYFQ GGGRMADFLK GLPVYNKSNF SRFHADSVCK ASNRRPSVYL PTREYPSEQI IVTEKTNILL RYLHQQWDKK NAAKKRDQEQ VELEGESSAP PRKVARTDSP DMHEDT

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.048 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClSodium chloridesodium chloride
2.0 mMTCEPtris(2-carboxyethyl)phosphine
20.0 uME7820N-(3-cyano-4-methyl-1H-indol-7-yl)-3-cyanobenzene-sulfonamide

Details: Gel filtration buffer was made fresh the day of the purification
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 286 K / Instrument: LEICA EM GP / Details: Blotted for 3 seconds before plunging..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.2 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 9393 / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 923678
Details: Initial 2D and 3D classification resulted in 923,678 particles
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1), RELION (ver. 3.0.4)) / Details: Standard CTF correction inside Relion
Startup modelType of model: OTHER
Details: Relion 3.0.4 was used to generate an initial model from the data.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.4) / Number images used: 75529
FSC plot (resolution estimation)

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