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- PDB-9hnw: USP1-UAF1 bound to Lys63-linked diubiquitin -

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Basic information

Entry
Database: PDB / ID: 9hnw
TitleUSP1-UAF1 bound to Lys63-linked diubiquitin
Components
  • (Polyubiquitin-B) x 2
  • Ubiquitin carboxyl-terminal hydrolase 1
  • WD repeat-containing protein 48
KeywordsHYDROLASE / USP1 / deubiquitinating enzyme / cysteine protease / complex
Function / homology
Function and homology information


regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / deubiquitinase activator activity / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / positive regulation of protein monoubiquitination / fat pad development / skeletal system morphogenesis ...regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / deubiquitinase activator activity / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / positive regulation of protein monoubiquitination / fat pad development / skeletal system morphogenesis / mitochondrion transport along microtubule / skin development / female gonad development / seminiferous tubule development / male meiosis I / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / homeostasis of number of cells / protein deubiquitination / embryonic organ development / single fertilization / regulation of DNA repair / response to UV / positive regulation of double-strand break repair via homologous recombination / energy homeostasis / regulation of neuron apoptotic process / neuron projection morphogenesis / regulation of proteasomal protein catabolic process / 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 / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / 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 / positive regulation of epithelial cell proliferation / 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 / skeletal system development / regulation of mitochondrial membrane potential / 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 / Ubiquitin-dependent degradation of Cyclin D / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / positive regulation of receptor signaling pathway via JAK-STAT
Similarity search - Function
WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase ...WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Papain-like cysteine peptidase superfamily / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / WD domain, G-beta repeat / Ubiquitin-like domain superfamily / G-protein beta WD-40 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
Ubiquitin carboxyl-terminal hydrolase 1 / Polyubiquitin-B / WD repeat-containing protein 48
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsKeijzer, N. / Sakoltchik, J. / Sixma, T.K.
Funding support Netherlands, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)TOP714.016.002 Netherlands
Health-HollandLSHM21048-H045 Netherlands
CitationJournal: Nat Commun / Year: 2025
Title: USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that can temporarily enrich for monoubiquitinated PCNA.
Authors: Niels Keijzer / Jan Sakoltchik / Kaustav Majumder / Nina van Lil / Farid El Oualid / Alexander Fish / Titia K Sixma /
Abstract: DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) ...DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as a substrate for further ubiquitination, forming K63-polyubiquitinated PCNA that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that formation of K48-linked chains also occurs on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about removal of ubiquitin chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis suggests that this mechanistic preference is maintained in evolution. This unusual mechanism can cause temporal enrichment of monoubiquitinated PCNA during polyubiquitination. It will be interesting to see how this affects DDT pathway balance.
History
DepositionDec 11, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 20, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquitin carboxyl-terminal hydrolase 1
B: WD repeat-containing protein 48
C: Polyubiquitin-B
D: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,1355
Polymers189,0694
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Ubiquitin carboxyl-terminal hydrolase 1 / Deubiquitinating enzyme 1 / hUBP / Ubiquitin thioesterase 1 / Ubiquitin-specific-processing protease 1


Mass: 91785.992 Da / Num. of mol.: 1 / Mutation: G670A, G671A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: USP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O94782, ubiquitinyl hydrolase 1
#2: Protein WD repeat-containing protein 48 / USP1-associated factor 1 / WD repeat endosomal protein / p80


Mass: 80130.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR48, KIAA1449, UAF1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TAF3
#3: Protein Polyubiquitin-B


Mass: 8604.884 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P0CG47
#4: Protein Polyubiquitin-B


Mass: 8547.770 Da / Num. of mol.: 1 / Mutation: K63(DAB) / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P0CG47
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
Has protein modificationY

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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: USP1-UAF1 bound to Lys63-linked diubiquitin / Type: COMPLEX / Entity ID: #1-#2, #4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesHepes1
2150 mMsodium chlorideNaCl1
32 mMTris(2-carboxyethyl)phosphine hydrochlorideTCEP1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Collected on Krios 1 at Netherlands Center for Electron Nanoscopy (NeCEN)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 10500 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.71 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5833

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Processing

EM software
IDName
1cryoSPARC
2PHENIX
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3110350
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 299304 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementHighest resolution: 3.04 Å

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