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- EMDB-62354: cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29-linked triU... -

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Entry
Database: EMDB / ID: EMD-62354
Titlecryo-EM structure of Ufd2/Ubc4-Ub in complex with K29-linked triUb (dimeric conformation)
Map data
Sample
  • Complex: Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb
    • Protein or peptide: Polyubiquitin-C
    • Protein or peptide: Ubiquitin
    • Protein or peptide: E4 ubiquitin-protein ligase UFD2
    • Protein or peptide: Ubiquitin-conjugating enzyme E2 4
KeywordsE4 enzyme / Ub ligase / Ufd2 / Ubc4 / Branching / K48/29 / LIGASE
Function / homology
Function and homology information


Peroxisomal protein import / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / mitochondria-associated ubiquitin-dependent protein catabolic process / cytoplasm protein quality control by the ubiquitin-proteasome system / E3 ubiquitin ligases ubiquitinate target proteins / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / ribosome-associated ubiquitin-dependent protein catabolic process / Formation of the ternary complex, and subsequently, the 43S complex / ubiquitin-ubiquitin ligase activity / E2 ubiquitin-conjugating enzyme ...Peroxisomal protein import / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / mitochondria-associated ubiquitin-dependent protein catabolic process / cytoplasm protein quality control by the ubiquitin-proteasome system / E3 ubiquitin ligases ubiquitinate target proteins / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / ribosome-associated ubiquitin-dependent protein catabolic process / Formation of the ternary complex, and subsequently, the 43S complex / ubiquitin-ubiquitin ligase activity / E2 ubiquitin-conjugating enzyme / Ribosomal scanning and start codon recognition / Translation initiation complex formation / proteasome binding / ubiquitin conjugating enzyme activity / SARS-CoV-1 modulates host translation machinery / cellular response to ethanol / Peptide chain elongation / Selenocysteine synthesis / Antigen processing: Ubiquitination & Proteasome degradation / Formation of a pool of free 40S subunits / Eukaryotic Translation Termination / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / Viral mRNA Translation / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / GTP hydrolysis and joining of the 60S ribosomal subunit / ubiquitin ligase complex / L13a-mediated translational silencing of Ceruloplasmin expression / Major pathway of rRNA processing in the nucleolus and cytosol / protein K48-linked ubiquitination / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / ERAD pathway / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Endosomal Sorting Complex Required For Transport (ESCRT) / Membrane binding and targetting of GAG proteins / Negative regulation of FLT3 / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Constitutive Signaling by NOTCH1 HD Domain Mutants / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / cytosolic ribosome / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / rescue of stalled ribosome / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / Regulation of FZD by ubiquitination / APC-Cdc20 mediated degradation of Nek2A / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / NF-kB is activated and signals survival / Regulation of innate immune responses to cytosolic DNA / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / NRIF signals cell death from the nucleus / VLDLR internalisation and degradation / Regulation of PTEN localization / Activated NOTCH1 Transmits Signal to the Nucleus / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / InlB-mediated entry of Listeria monocytogenes into host cell / Translesion synthesis by POLK / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Josephin domain DUBs / Translesion synthesis by POLI / IKK complex recruitment mediated by RIP1 / Regulation of activated PAK-2p34 by proteasome mediated degradation / ubiquitin binding / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of protein ubiquitination / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / TCF dependent signaling in response to WNT / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Regulation of NF-kappa B signaling / Asymmetric localization of PCP proteins / activated TAK1 mediates p38 MAPK activation
Similarity search - Function
Ubiquitin conjugation factor E4, core / Ubiquitin conjugation factor E4 / Ubiquitin elongating factor core / U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 ...Ubiquitin conjugation factor E4, core / Ubiquitin conjugation factor E4 / Ubiquitin elongating factor core / U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / S27a-like superfamily / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc-binding ribosomal protein / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Polyubiquitin-C / Ubiquitin-conjugating enzyme E2 4 / E4 ubiquitin-protein ligase UFD2 / Ubiquitin-ribosomal protein eS31 fusion protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Homo sapiens (human) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.85 Å
AuthorsAi HS / Tong ZB / Liu L
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2137005, 92253302, 22227810, 22407121, T2488301, 22277073 and 92253302 China
CitationJournal: Nat Chem Biol / Year: 2025
Title: Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains.
Authors: Zebin Tong / Xiangwei Wu / Hongyi Cai / Shidian Wu / Tianyi Zhang / Zhiheng Deng / Ziyu Xu / Rujing Yuan / Huasong Ai / Lei Liu / Man Pan /
Abstract: E4 enzymes amplify and remodel ubiquitin chain signals beyond the conventional E1-E2-E3 cascade. The first identified E4 enzyme Ufd2 preferentially catalyzes K48/K29 branched ubiquitin chains, yet ...E4 enzymes amplify and remodel ubiquitin chain signals beyond the conventional E1-E2-E3 cascade. The first identified E4 enzyme Ufd2 preferentially catalyzes K48/K29 branched ubiquitin chains, yet the structural mechanism remains unknown. Here, we combined chemical biology and cryo-electron microscopy to visualize stable intermediates in Ufd2 loading ubiquitin at K48 of proximal ubiquitin on K29-linked di- and triubiquitin. Our data reveal that the core region of Ufd2 functions as an unprecedented K29 diubiquitin binding domain, interacting extensively with proximal and distal ubiquitin, which orients the K48 site of proximal ubiquitin toward the active site of Ubc4, facilitating K48/K29 branched ubiquitin chain formation. We also identified a unique dimeric conformation where dimerized Ufd2 and Ubc4 stabilize each other's distal ubiquitin during branching on K29 triubiquitin. Our findings provide mechanistic insights into the assembly of K48/K29 branched ubiquitin chains by the E4 enzyme Ufd2 and highlight the spatial cooperation among multiple pairs of ubiquitin-related enzymes on longer ubiquitin chains.
History
DepositionNov 11, 2024-
Header (metadata) releaseJul 30, 2025-
Map releaseJul 30, 2025-
UpdateAug 27, 2025-
Current statusAug 27, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62354.png

Downloads & links

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Map

FileDownload / File: emd_62354.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 281.062 Å		1.1 Å/pix.
x 256 pix.
= 281.062 Å		1.1 Å/pix.
x 256 pix.
= 281.062 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0979 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.05530696 - 0.08576865
Average (Standard dev.)0.000027051838 (±0.0035128724)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.0624 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62354_msk_1.map
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Additional map: #1

Fileemd_62354_additional_1.map
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Half map: #2

Fileemd_62354_half_map_1.map
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Half map: #1

Fileemd_62354_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb

EntireName: Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb
Components
  • Complex: Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb
    • Protein or peptide: Polyubiquitin-C
    • Protein or peptide: Ubiquitin
    • Protein or peptide: E4 ubiquitin-protein ligase UFD2
    • Protein or peptide: Ubiquitin-conjugating enzyme E2 4

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Supramolecule #1: Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb

SupramoleculeName: Cryo-EM structure of Ufd2/Ubc4-Ub in complex with K29triUb
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Polyubiquitin-C

MacromoleculeName: Polyubiquitin-C / type: protein_or_peptide / ID: 1 / Details: K29triUb (proximal Ub) / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.550794 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGG

UniProtKB: Polyubiquitin-C

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Macromolecule #2: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.576831 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

UniProtKB: Ubiquitin-ribosomal protein eS31 fusion protein

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Macromolecule #3: E4 ubiquitin-protein ligase UFD2

MacromoleculeName: E4 ubiquitin-protein ligase UFD2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 109.360742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTAIEDILQI TTDPSDTRGY SLLKSEEVPQ GSTLGVDFID TLLLYQLTEN EKLDKPFEYL NDCFRRNQQQ KRITKNKPNA ESLHSTFQE IDRLVIGYGV VALQIENFCM NGAFINYITG IVSNVNSYTD FLSQIIQRAI LEGTALDLLN AVFPTLLEYC N KHVSHFDL ...String:
MTAIEDILQI TTDPSDTRGY SLLKSEEVPQ GSTLGVDFID TLLLYQLTEN EKLDKPFEYL NDCFRRNQQQ KRITKNKPNA ESLHSTFQE IDRLVIGYGV VALQIENFCM NGAFINYITG IVSNVNSYTD FLSQIIQRAI LEGTALDLLN AVFPTLLEYC N KHVSHFDL NESVIYNNVL TIFELFVTFK PIAEIFTKID GFFADYSCKP QDFERKTILG PILSLSPIEA AVAIRNYGDN LL RSKQQTA MIHESLQAEH KVVIDRLFFI VDKLVRGSLN SRTDMISYFA HIANKNHLRR ADHPPFKELS SNGFMSNITL LLV RFSQPF LDISYKKIDK IDANYFNNPS LFIDLSGETR LNSDFKEADA FYDKNRKTAD SKPNFISDCF FLTLTYLHYG LGGT LSFEE KMGSEIKALK EEIEKVKKIA ANHDVFARFI TAQLSKMEKA LKTTESLRFA LQGFFAHRSL QLEVFDFICG ASTFL IRVV DPEHEFPFKQ IKLPLIPDQI GVENVDNADF LRAHAPVPFK YYPEFVVEGP VNYSLYISKY QTSPIFRNPR LGSFVE FTT MVLRCPELVS NPHLKGKLVQ LLSVGAMPLT DNSPGFMMDI FEHDELVNKN LLYALLDFYV IVEKTGSSSQ FYDKFNS RY SISIILEELY YKIPSYKNQL IWQSQNNADF FVRFVARMLN DLTFLLDEGL SNLAEVHNIQ NELDNRARGA PPTREEED K ELQTRLASAS RQAKSSCGLA DKSMKLFEIY SKDIPAAFVT PEIVYRLASM LNYNLESLVG PKCGELKVKD PQSYSFNPK DLLKALTTVY INLSEQSEFI SAVAKDERSF NRNLFVRAVD ILGRKTGLAS PEFIEKLLNF ANKAEEQRKA DEEEDLEYGD VPDEFLDPL MYTIMKDPVI LPASKMNIDR STIKAHLLSD STDPFNRMPL KLEDVTPNEE LRQKILCFKK QKKEEA

UniProtKB: E4 ubiquitin-protein ligase UFD2

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Macromolecule #4: Ubiquitin-conjugating enzyme E2 4

MacromoleculeName: Ubiquitin-conjugating enzyme E2 4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 16.442586 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSSSKRIAKE LSDLERDPPT SSSAGPVGDD LYHWQASIMG PADSPYAGGV FFLSIHFPTD YPFKPPKISF TTKIYHPNIN ANGNICLDI LKDQWSPALT LSKVLLSISS LLTDANPDDP LVPEIAHIYK TDRPKYEATA REWTKKYAV

UniProtKB: Ubiquitin-conjugating enzyme E2 4

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48544
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION

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