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- EMDB-45226: Cryo EM structure of a DCAF2:degrader:BRD4 ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-45226
TitleCryo EM structure of a DCAF2:degrader:BRD4 ternary complex
Map data
Sample
  • Complex: DCAF2:degrader:BRD4 ternary complex
    • Protein or peptide: Denticleless protein homolog
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: DET1- and DDB1-associated protein 1
    • Protein or peptide: Bromodomain-containing protein 4
  • Ligand: N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cyclopentane-1,2'-quinoxalin]-6'-yl]oxy}piperidin-1-yl)methyl]phenyl}methyl)-2-[(6S,10P)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetamide
KeywordsDTL / DDB1 / DDA1 / BRD4 / degrader / covalent / LIGASE
Function / homology
Function and homology information


positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex ...positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / UV-damage excision repair / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / mitotic G2 DNA damage checkpoint signaling / negative regulation of reproductive process / negative regulation of developmental process / RNA polymerase II C-terminal domain binding / cullin family protein binding / P-TEFb complex binding / viral release from host cell / negative regulation of DNA damage checkpoint / histone H4 reader activity / host-mediated suppression of viral transcription / protein monoubiquitination / positive regulation of G2/M transition of mitotic cell cycle / ectopic germ cell programmed cell death / translesion synthesis / positive regulation of T-helper 17 cell lineage commitment / positive regulation of viral genome replication / response to UV / proteasomal protein catabolic process / : / positive regulation of gluconeogenesis / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome / transcription coregulator activity / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / Recognition of DNA damage by PCNA-containing replication complex / regulation of circadian rhythm / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Formation of Incision Complex in GG-NER / Wnt signaling pathway / protein polyubiquitination / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / cellular response to UV / p53 binding / rhythmic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / chromosome / site of double-strand break / Neddylation / regulation of inflammatory response / ubiquitin-dependent protein catabolic process / nuclear membrane / protein-macromolecule adaptor activity / histone binding / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / transcription coactivator activity / chromosome, telomeric region / DNA replication / positive regulation of canonical NF-kappaB signal transduction / transcription cis-regulatory region binding / regulation of cell cycle / protein ubiquitination / chromatin remodeling / DNA repair / protein serine/threonine kinase activity / apoptotic process / DNA damage response / centrosome / chromatin binding / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / chromatin / positive regulation of DNA-templated transcription / protein-containing complex binding / nucleolus / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / extracellular space / DNA binding / extracellular exosome / nucleoplasm / nucleus / cytoplasm / cytosol
Similarity search - Function
: / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller ...: / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / Brdt, bromodomain, repeat I / Brdt, bromodomain, repeat II / NET domain superfamily / NET domain profile. / : / NET domain / Bromodomain extra-terminal - transcription regulation / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / WD domain, G-beta repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Bromodomain-containing protein 4 / DNA damage-binding protein 1 / DET1- and DDB1-associated protein 1 / Denticleless protein homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsMcMahon EJ / Wang W
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2025
Title: Structural basis for DCAF2 as a novel E3 ligase for PROTAC-mediated targeted protein degradation.
Authors: Evan J McMahon / Alexander G Cioffi / Patrick R Visperas / Yueqing Lin / Michael Shaghafi / Courtney M Daczkowski / Johannes C Hermann / Robert A Everley / Richard M Neve / Daniel A Erlanson ...Authors: Evan J McMahon / Alexander G Cioffi / Patrick R Visperas / Yueqing Lin / Michael Shaghafi / Courtney M Daczkowski / Johannes C Hermann / Robert A Everley / Richard M Neve / Daniel A Erlanson / Kevin R Webster / Vikram Narayan / Weiru Wang /
Abstract: Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 ...Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 human E3 ligases. To expand this repertoire, we conducted structural and functional validation of DDB1 (Damage-specific DNA binding protein 1) and Cullin-associated factor (DCAF)2 (DTL/CDT2), a Cullin4-RING ligase substrate adaptor implicated in DNA damage response and cancer, as a novel E3 for TPD. Cryoelectron microscopy (cryo-EM) structures of the DCAF2:DDB1:DDA1 complex (3.3 Å), a ligand bound complex (3.1 Å), and a ternary complex with a covalent proteolysis-targeting chimera (PROTAC) and BRD4 (3.4 Å) reveal PROTAC-mediated substrate recruitment. Using covalent bifunctional tool compounds engaging residue C141 in the WD40 domain, we demonstrate robust ubiquitination in biochemical assays and cellular TPD using the COFFEE (covalent functionalization followed by E3 electroporation) method. These findings position DCAF2 as a promising E3 adaptor for PROTAC strategies and identify C141 as a relevant site for future PROTAC discovery.
History
DepositionJun 6, 2024-
Header (metadata) releaseOct 1, 2025-
Map releaseOct 1, 2025-
UpdateOct 15, 2025-
Current statusOct 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45226.png

Downloads & links

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Map

FileDownload / File: emd_45226.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.003896
Minimum - Maximum-0.02309389 - 0.07242864
Average (Standard dev.)0.00021709822 (±0.0017561123)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45226_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45226_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : DCAF2:degrader:BRD4 ternary complex

EntireName: DCAF2:degrader:BRD4 ternary complex
Components
  • Complex: DCAF2:degrader:BRD4 ternary complex
    • Protein or peptide: Denticleless protein homolog
    • Protein or peptide: DNA damage-binding protein 1
    • Protein or peptide: DET1- and DDB1-associated protein 1
    • Protein or peptide: Bromodomain-containing protein 4
  • Ligand: N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cyclopentane-1,2'-quinoxalin]-6'-yl]oxy}piperidin-1-yl)methyl]phenyl}methyl)-2-[(6S,10P)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetamide

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Supramolecule #1: DCAF2:degrader:BRD4 ternary complex

SupramoleculeName: DCAF2:degrader:BRD4 ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4, #3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 206 KDa

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Macromolecule #1: Denticleless protein homolog

MacromoleculeName: Denticleless protein homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.887121 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLFNSVLRQP QLGVLRNGWS SQYPLQSLLT GYQCSGNDEH TSYGETGVPV PPFGCTFSSA PNMEHVLAVA NEEGFVRLYN TESQSFRKK CFKEWMAHWN AVFDLAWVPG ELKLVTAAGD QTAKFWDVKA GELIGTCKGH QCSLKSVAFS KFEKAVFCTG G RDGNIMVW ...String:
MLFNSVLRQP QLGVLRNGWS SQYPLQSLLT GYQCSGNDEH TSYGETGVPV PPFGCTFSSA PNMEHVLAVA NEEGFVRLYN TESQSFRKK CFKEWMAHWN AVFDLAWVPG ELKLVTAAGD QTAKFWDVKA GELIGTCKGH QCSLKSVAFS KFEKAVFCTG G RDGNIMVW DTRCNKKDGF YRQVNQISGA HNTSDKQTPS KPKKKQNSKG LAPSVDFQQS VTVVLFQDEN TLVSAGAVDG II KVWDLRK NYTAYRQEPI ASKSFLYPGS STRKLGYSSL ILDSTGSTLF ANCTDDNIYM FNMTGLKTSP VAIFNGHQNS TFY VKSSLS PDDQFLVSGS SDEAAYIWKV STPWQPPTVL LGHSQEVTSV CWCPSDFTKI ATCSDDNTLK IWRLNRGLEE KPGG DKLST VGWASQKKKE SRPGLVTVTS SQSTPAKAPR AKCNPSNSSP SSAACAPSCA GDLENLYFQS HHHHHH

UniProtKB: Denticleless protein homolog

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Macromolecule #2: DNA damage-binding protein 1

MacromoleculeName: DNA damage-binding protein 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 127.097469 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYNYVVTAQ KPTAVNGCVT GHFTSAEDLN LLIAKNTRLE IYVVTAEGLR PVKEVGMYGK IAVMELFRPK GESKDLLFIL TAKYNACIL EYKQSGESID IITRAHGNVQ DRIGRPSETG IIGIIDPECR MIGLRLYDGL FKVIPLDRDN KELKAFNIRL E ELHVIDVK ...String:
MSYNYVVTAQ KPTAVNGCVT GHFTSAEDLN LLIAKNTRLE IYVVTAEGLR PVKEVGMYGK IAVMELFRPK GESKDLLFIL TAKYNACIL EYKQSGESID IITRAHGNVQ DRIGRPSETG IIGIIDPECR MIGLRLYDGL FKVIPLDRDN KELKAFNIRL E ELHVIDVK FLYGCQAPTI CFVYQDPQGR HVKTYEVSLR EKEFNKGPWK QENVEAEASM VIAVPEPFGG AIIIGQESIT YH NGDKYLA IAPPIIKQST IVCHNRVDPN GSRYLLGDME GRLFMLLLEK EEQMDGTVTL KDLRVELLGE TSIAECLTYL DNG VVFVGS RLGDSQLVKL NVDSNEQGSY VVAMETFTNL GPIVDMCVVD LERQGQGQLV TCSGAFKEGS LRIIRNGIGI HEHA SIDLP GIKGLWPLRS DPNRETDDTL VLSFVGQTRV LMLNGEEVEE TELMGFVDDQ QTFFCGNVAH QQLIQITSAS VRLVS QEPK ALVSEWKEPQ AKNISVASCN SSQVVVAVGR ALYYLQIHPQ ELRQISHTEM EHEVACLDIT PLGDSNGLSP LCAIGL WTD ISARILKLPS FELLHKEMLG GEIIPRSILM TTFESSHYLL CALGDGALFY FGLNIETGLL SDRKKVTLGT QPTVLRT FR SLSTTNVFAC SDRPTVIYSS NHKLVFSNVN LKEVNYMCPL NSDGYPDSLA LANNSTLTIG TIDEIQKLHI RTVPLYES P RKICYQEVSQ CFGVLSSRIE VQDTSGGTTA LRPSASTQAL SSSVSSSKLF SSSTAPHETS FGEEVEVHNL LIIDQHTFE VLHAHQFLQN EYALSLVSCK LGKDPNTYFI VGTAMVYPEE AEPKQGRIVV FQYSDGKLQT VAEKEVKGAV YSMVEFNGKL LASINSTVR LYEWTTEKEL RTECNHYNNI MALYLKTKGD FILVGDLMRS VLLLAYKPME GNFEEIARDF NPNWMSAVEI L DDDNFLGA ENAFNLFVCQ KDSAATTDEE RQHLQEVGLF HLGEFVNVFC HGSLVMQNLG ETSTPTQGSV LFGTVNGMIG LV TSLSESW YNLLLDMQNR LNKVIKSVGK IEHSFWRSFH TERKTEPATG FIDGDLIESF LDISRPKMQE VVANLQYDDG SGM KREATA DDLIKVVEEL TRIH

UniProtKB: DNA damage-binding protein 1

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Macromolecule #3: Bromodomain-containing protein 4

MacromoleculeName: Bromodomain-containing protein 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.568838 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MHHHHHHETS NPNKPKRQTN QLQYLLRVVL KTLWKHQFAW PFQQPVDAVK LNLPDYYKII KTPMDMGTIK KRLENNYYWN AQECIQDFN TMFTNCYIYN KPGDDIVLMA EALEKLFLQK INELPTEETE

UniProtKB: Bromodomain-containing protein 4

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Macromolecule #4: DET1- and DDB1-associated protein 1

MacromoleculeName: DET1- and DDB1-associated protein 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.855297 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MADFLKGLPV YNKSNFSRFH ADSVCKASNR RPSVYLPTRE YPSEQIIVTE KTNILLRYLH QQWDKKNAAK KRDQEQVELE GESSAPPRK VARTDSPDMH EDT

UniProtKB: DET1- and DDB1-associated protein 1

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Macromolecule #5: N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cycl...

MacromoleculeName: N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cyclopentane-1,2'-quinoxalin]-6'-yl]oxy}piperidin-1-yl)methyl]phenyl}methyl)-2-[(6S,10P)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- ...Name: N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cyclopentane-1,2'-quinoxalin]-6'-yl]oxy}piperidin-1-yl)methyl]phenyl}methyl)-2-[(6S,10P)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: A1AUO
Molecular weightTheoretical: 879.896 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
SoftwareName: Leginon
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 1.4 sec. / Average electron dose: 50.21 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1277208
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 70550
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: Coot
Output model

PDB-9c5v:
Cryo EM structure of a DCAF2:degrader:BRD4 ternary complex

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