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- PDB-7ay1: Cryo-EM structure of USP1-UAF1 bound to mono-ubiquitinated FANCD2... -

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Basic information

Entry
Database: PDB / ID: 7ay1
TitleCryo-EM structure of USP1-UAF1 bound to mono-ubiquitinated FANCD2, and FANCI
Components
  • (Fanconi anemia group ...) x 2
  • DNA (61-MER)
  • Ubiquitin carboxyl-terminal hydrolase 1
  • Ubiquitin-60S ribosomal protein L40
  • WD repeat-containing protein 48
KeywordsHYDROLASE / deubiquitination / specificity / DNA repair / Fanconi Anemia
Function / homology
Function and homology information


positive regulation of error-prone translesion synthesis / regulation of protein monoubiquitination / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / regulation of CD40 signaling pathway / regulation of regulatory T cell differentiation / monoubiquitinated protein deubiquitination / homologous chromosome pairing at meiosis / double-strand break repair involved in meiotic recombination / gamete generation / neuronal stem cell population maintenance ...positive regulation of error-prone translesion synthesis / regulation of protein monoubiquitination / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / regulation of CD40 signaling pathway / regulation of regulatory T cell differentiation / monoubiquitinated protein deubiquitination / homologous chromosome pairing at meiosis / double-strand break repair involved in meiotic recombination / gamete generation / neuronal stem cell population maintenance / deubiquitinase activator activity / brain morphogenesis / DNA repair complex / mitotic intra-S DNA damage checkpoint signaling / skeletal system morphogenesis / skin development / positive regulation of double-strand break repair via homologous recombination / seminiferous tubule development / protein deubiquitination / single fertilization / Peptide chain elongation / Selenocysteine synthesis / 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) / homeostasis of number of cells / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / Major pathway of rRNA processing in the nucleolus and cytosol / embryonic organ development / regulation of DNA repair / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / interstrand cross-link repair / DNA polymerase binding / response to UV / condensed chromosome / 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 / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / Endosomal Sorting Complex Required For Transport (ESCRT) / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Regulation of FZD by ubiquitination / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / 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 / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / cytosolic ribosome / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / ubiquitin binding / APC/C:Cdc20 mediated degradation of Securin / positive regulation of protein ubiquitination / Asymmetric localization of PCP proteins
Similarity search - Function
Ubiquitin specific peptidase 1 / WDR48/Bun107 / Domain of unknown function (DUF3337) / Fanconi anemia group I protein / FANCI solenoid 1 cap / FANCI solenoid 1 domain / FANCI helical domain 1 / FANCI helical domain 2 / FANCI solenoid 3 domain / FANCI solenoid 4 domain ...Ubiquitin specific peptidase 1 / WDR48/Bun107 / Domain of unknown function (DUF3337) / Fanconi anemia group I protein / FANCI solenoid 1 cap / FANCI solenoid 1 domain / FANCI helical domain 1 / FANCI helical domain 2 / FANCI solenoid 3 domain / FANCI solenoid 4 domain / FANCI solenoid 2 domain / FANCI solenoid 1 cap / FANCI solenoid 1 / FANCI solenoid 2 / FANCI solenoid 3 / FANCI solenoid 4 / FANCI helical domain 1 / FANCI helical domain 2 / Fanconi anaemia protein FANCD2 / Fanconi anaemia protein FancD2 nuclease / 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. / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Papain-like cysteine peptidase superfamily / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / G-protein beta WD-40 repeat / Ubiquitin-like domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Ubiquitin carboxyl-terminal hydrolase 1 / Ubiquitin-ribosomal protein eL40 fusion protein / WD repeat-containing protein 48 / Fanconi anemia group D2 protein / Fanconi anemia group I protein
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRennie, M.L. / Arkinson, C. / Walden, H.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-2015-CoG-681582 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UU_12016/12 United Kingdom
Citation
Journal: Nat Struct Mol Biol / Year: 2021
Title: Structural basis of FANCD2 deubiquitination by USP1-UAF1.
Authors: Martin L Rennie / Connor Arkinson / Viduth K Chaugule / Rachel Toth / Helen Walden /
Abstract: Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the ...Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.
#1: Journal: Biorxiv / Year: 2020
Title: Structural basis of FANCD2 deubiquitination by USP1-UAF1
Authors: Rennie, M.L. / Arkinson, C. / Chaugule, V.K. / Toth, R. / Walden, H.
History
DepositionNov 10, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 24, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 7, 2021Group: Database references / Category: citation / citation_author
Revision 1.2Apr 14, 2021Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Apr 28, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Jul 27, 2022Group: Advisory / Database references ...Advisory / Database references / Polymer sequence / Source and taxonomy / Structure summary
Category: database_2 / entity ...database_2 / entity / entity_poly / entity_poly_seq / entity_src_gen / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.formula_weight / _entity.pdbx_mutation / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_poly_seq.mon_id / _entity_src_gen.gene_src_common_name / _pdbx_poly_seq_scheme.mon_id / _pdbx_unobs_or_zero_occ_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Fanconi anemia group I protein
B: Fanconi anemia group D2 protein
C: Ubiquitin-60S ribosomal protein L40
D: Ubiquitin carboxyl-terminal hydrolase 1
E: WD repeat-containing protein 48
S: DNA (61-MER)
T: DNA (61-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)501,0708
Polymers501,0057
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, USP1, UAF1, FANCI, and FANCD2ub all coelute
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Fanconi anemia group ... , 2 types, 2 molecules AB

#1: Protein Fanconi anemia group I protein / Protein FACI


Mass: 150459.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FANCI, KIAA1794 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NVI1
#2: Protein Fanconi anemia group D2 protein / Protein FACD2


Mass: 164623.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FANCD2, FACD / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BXW9

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Protein , 3 types, 3 molecules CDE

#3: Protein Ubiquitin-60S ribosomal protein L40 / CEP52 / Ubiquitin A-52 residue ribosomal protein fusion product 1


Mass: 8875.125 Da / Num. of mol.: 1 / Mutation: GPGS expression tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBA52, UBCEP2 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62987
#4: Protein Ubiquitin carboxyl-terminal hydrolase 1 / Deubiquitinating enzyme 1 / hUBP / Ubiquitin thioesterase 1 / Ubiquitin-specific-processing protease 1


Mass: 88390.273 Da / Num. of mol.: 1 / Mutation: C90S, G670A, G671A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: USP1 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O94782, ubiquitinyl hydrolase 1
#5: Protein WD repeat-containing protein 48 / USP1-associated factor 1 / WD repeat endosomal protein / p80


Mass: 78300.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR48, KIAA1449, UAF1 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TAF3

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DNA chain / Non-polymers , 2 types, 3 molecules ST

#6: DNA chain DNA (61-MER)


Mass: 5177.820 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Arbitrary DNA sequence modelled due to insufficient local resolution to determine sequence register. DNA used TGATCAGAGGTCATTTGAATTCATGGCTTCGAGCTTCATGTAGAGTCGACGGTGCTGGGAT
Source: (synth.) synthetic construct (others)
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Enzyme substrate complex of USP1-UAF1 and FANCI-FANCD2ub-dsDNACOMPLEX#1-#60MULTIPLE SOURCES
2Enzyme substrate complex of USP1-UAF1 and FANCI-FANCD2ubCOMPLEX#1-#51RECOMBINANT
3DNACOMPLEX#61RECOMBINANT
Molecular weightValue: 0.49 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33synthetic construct (others)32630
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMNaClSodium chlorideNaClSodium chloride1
32 mMDTTC4H10O2S21
SpecimenConc.: 4.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 14.9 sec. / Electron dose: 65 e/Å2 / Detector mode: COUNTING / Film or detector model: DIRECT ELECTRON DE-64 (8k x 8k)
Image scansMovie frames/image: 59

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Processing

SoftwareName: PHENIX / Version: (1.18_3855:phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
4cryoSPARC2.13.2CTF correction
13cryoSPARC2.13.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 249732
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 391552 / Symmetry type: POINT
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 370.07 Å2 / Biso mean: 65.7212 Å2 / Biso min: 17.11 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00526319
ELECTRON MICROSCOPYf_angle_d0.85435840
ELECTRON MICROSCOPYf_chiral_restr0.0484209
ELECTRON MICROSCOPYf_plane_restr0.0054301
ELECTRON MICROSCOPYf_dihedral_angle_d20.3433822

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