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- PDB-8a9k: Cryo-EM structure of USP1-UAF1 bound to FANCI and mono-ubiquitina... -

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Basic information

Entry
Database: PDB / ID: 8a9k
TitleCryo-EM structure of USP1-UAF1 bound to FANCI and mono-ubiquitinated FANCD2 with ML323 (consensus reconstruction)
Components
  • (Fanconi anemia group ...) x 2
  • DNA (61-MER)
  • Polyubiquitin-C
  • Ubiquitin carboxyl-terminal hydrolase 1
  • WD repeat-containing protein 48
KeywordsHYDROLASE / Deubiquitinase / Complex / Enzyme-Substrate / Inhibitor
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 / regulation of CD40 signaling pathway / regulation of regulatory T cell differentiation / gamete generation / double-strand break repair involved in meiotic recombination / monoubiquitinated protein deubiquitination / homologous chromosome pairing at meiosis / neuronal stem cell population maintenance ...regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / regulation of CD40 signaling pathway / regulation of regulatory T cell differentiation / gamete generation / double-strand break repair involved in meiotic recombination / monoubiquitinated protein deubiquitination / homologous chromosome pairing at meiosis / neuronal stem cell population maintenance / brain morphogenesis / deubiquitinase activator activity / mitotic intra-S DNA damage checkpoint signaling / DNA repair complex / skeletal system morphogenesis / skin development / seminiferous tubule development / homeostasis of number of cells / protein deubiquitination / single fertilization / embryonic organ development / positive regulation of double-strand break repair via homologous recombination / interstrand cross-link repair / regulation of DNA repair / response to UV / condensed chromosome / DNA polymerase binding / 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 / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Constitutive Signaling by NOTCH1 HD Domain Mutants / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / 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 / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / APC-Cdc20 mediated degradation of Nek2A / 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 / Regulation of innate immune responses to cytosolic DNA / NF-kB is activated and signals survival / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / NRIF signals cell death from the nucleus / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / Downregulation of TGF-beta receptor signaling / positive regulation of epithelial cell proliferation / Josephin domain DUBs / ubiquitin binding / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Regulation of activated PAK-2p34 by proteasome mediated degradation / Translesion synthesis by POLI / IKK complex recruitment mediated by RIP1 / positive regulation of protein ubiquitination / Gap-filling DNA repair synthesis and ligation in GG-NER / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / skeletal system development / 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 / response to gamma radiation / positive regulation of receptor signaling pathway via JAK-STAT / Regulation of NF-kappa B signaling / Asymmetric localization of PCP proteins / Ubiquitin-dependent degradation of Cyclin D / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / activated TAK1 mediates p38 MAPK activation
Similarity search - Function
WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / 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 ...WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / 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. / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Papain-like cysteine peptidase superfamily / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin-like (UB roll) / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / 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 / Roll / Alpha Beta
Similarity search - Domain/homology
Chem-JDA / DNA / DNA (> 10) / Ubiquitin carboxyl-terminal hydrolase 1 / Polyubiquitin-C / 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: 2.85 Å
AuthorsRennie, M.L. / Walden, H.
Funding supportEuropean Union, United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-2015-CoG-681582European Union
Medical Research Council (MRC, United Kingdom)MC_UU_12016/12 United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM reveals a mechanism of USP1 inhibition through a cryptic binding site.
Authors: Martin L Rennie / Connor Arkinson / Viduth K Chaugule / Helen Walden /
Abstract: Repair of DNA damage is critical to genomic integrity and frequently disrupted in cancers. Ubiquitin-specific protease 1 (USP1), a nucleus-localized deubiquitinase, lies at the interface of multiple ...Repair of DNA damage is critical to genomic integrity and frequently disrupted in cancers. Ubiquitin-specific protease 1 (USP1), a nucleus-localized deubiquitinase, lies at the interface of multiple DNA repair pathways and is a promising drug target for certain cancers. Although multiple inhibitors of this enzyme, including one in phase 1 clinical trials, have been established, their binding mode is unknown. Here, we use cryo-electron microscopy to study an assembled enzyme-substrate-inhibitor complex of USP1 and the well-established inhibitor, ML323. Achieving 2.5-Å resolution, with and without ML323, we find an unusual binding mode in which the inhibitor disrupts part of the hydrophobic core of USP1. The consequent conformational changes in the secondary structure lead to subtle rearrangements in the active site that underlie the mechanism of inhibition. These structures provide a platform for structure-based drug design targeting USP1.
History
DepositionJun 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 2.0Mar 1, 2023Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / em_entity_assembly ...atom_site / em_entity_assembly / entity / entity_poly / entity_poly_seq / pdbx_entity_nonpoly / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_conn_angle / struct_conn / struct_ref / struct_ref_seq
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site.type_symbol / _em_entity_assembly.entity_id_list / _entity.formula_weight / _entity.pdbx_description / _entity.type / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _struct_conn.ptnr2_label_asym_id
Revision 2.1Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature
Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fanconi anemia group I protein
B: Fanconi anemia group D2 protein
C: Polyubiquitin-C
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)501,4559
Polymers501,0057
Non-polymers4502
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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 Polyubiquitin-C


Mass: 8875.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0CG48
#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
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 , 1 types, 2 molecules ST

#6: DNA chain DNA (61-MER)


Mass: 5177.820 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Actual sequence: TGATCAGAGGTCATTTGAATTCATGGCTTCGAGCTTCATGTAGAGTCGACGGTGCTGGGAT
Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-JDA / 5-methyl-2-(2-propan-2-ylphenyl)-~{N}-[[4-(1,2,3-triazol-1-yl)phenyl]methyl]pyrimidin-4-amine


Mass: 384.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H24N6 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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(C90S)-UAF1 bound to FANCI and mono-ubiquitinated FANCD2 with dsDNA and ML323
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 9.3 uM USP1-UAF1, 1.8 uM FANCI-FANCD2Ub, 2.2 uM dsDNA (61 base-pairs), 18 uM ML323
Specimen supportGrid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
4cryoSPARCCTF correction
7Coot0.9.6model fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIX1.19model refinement
Image processingDetails: 2x binning
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6716868
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 570816 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 95.3 / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00324982
ELECTRON MICROSCOPYf_angle_d0.53634012
ELECTRON MICROSCOPYf_dihedral_angle_d16.669392
ELECTRON MICROSCOPYf_chiral_restr0.0423988
ELECTRON MICROSCOPYf_plane_restr0.0034084

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