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- PDB-9r85: Cryo-EM structure of the E3 ligase HECTD3 conjugated to ubiquitin -

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Entry
Database: PDB / ID: 9r85
TitleCryo-EM structure of the E3 ligase HECTD3 conjugated to ubiquitin
Components
  • E3 ubiquitin-protein ligase HECTD3
  • Ubiquitin
KeywordsLIGASE / E3-ligase / ubiquitin / ubiquitination / HECT-ligase
Function / homology
Function and homology information


HECT-type E3 ubiquitin transferase / syntaxin binding / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / IRAK2 mediated activation of TAK1 complex / Prevention of phagosomal-lysosomal fusion / Alpha-protein kinase 1 signaling pathway ...HECT-type E3 ubiquitin transferase / syntaxin binding / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / IRAK2 mediated activation of TAK1 complex / Prevention of phagosomal-lysosomal fusion / 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 / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / 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 / Regulation of pyruvate metabolism / NF-kB is activated and signals survival / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Pexophagy / Regulation of innate immune responses to cytosolic DNA / Downregulation of ERBB2:ERBB3 signaling / 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 / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / Translesion synthesis by POLI / IKK complex recruitment mediated by RIP1 / Regulation of activated PAK-2p34 by proteasome mediated degradation / 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 / Autodegradation of Cdh1 by Cdh1:APC/C / TCF dependent signaling in response to WNT / APC/C:Cdc20 mediated degradation of Securin / Regulation of NF-kappa B signaling / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / activated TAK1 mediates p38 MAPK activation / Asymmetric localization of PCP proteins / Ubiquitin-dependent degradation of Cyclin D / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Regulation of signaling by CBL / TNFR2 non-canonical NF-kB pathway / AUF1 (hnRNP D0) binds and destabilizes mRNA / NOTCH3 Activation and Transmission of Signal to the Nucleus / Negative regulators of DDX58/IFIH1 signaling / Assembly of the pre-replicative complex / Negative regulation of FGFR3 signaling / Fanconi Anemia Pathway / Peroxisomal protein import / Vpu mediated degradation of CD4 / Deactivation of the beta-catenin transactivating complex / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Stabilization of p53 / Degradation of DVL / Negative regulation of FGFR2 signaling / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of CRY and PER proteins / Negative regulation of FGFR4 signaling / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Degradation of AXIN / Negative regulation of FGFR1 signaling / Regulation of TNFR1 signaling / EGFR downregulation / Termination of translesion DNA synthesis / Hh mutants are degraded by ERAD / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / Activation of NF-kappaB in B cells / Assembly Of The HIV Virion / G2/M Checkpoints / Hedgehog ligand biogenesis / Degradation of GLI1 by the proteasome
Similarity search - Function
E3 ubiquitin-protein ligase HECTD3 / APC10/DOC domain / Anaphase-promoting complex, subunit 10 (APC10) / DOC domain profile. / Anaphase-promoting complex, subunit 10 (APC10) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with ...E3 ubiquitin-protein ligase HECTD3 / APC10/DOC domain / Anaphase-promoting complex, subunit 10 (APC10) / DOC domain profile. / Anaphase-promoting complex, subunit 10 (APC10) / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Galactose-binding-like domain superfamily / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
prop-2-en-1-amine / Polyubiquitin-C / E3 ubiquitin-protein ligase HECTD3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsEsposito, D. / Huber, J. / Maslen, S. / Rittinger, K.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
The Francis Crick InstituteCC2075, CC2000 United Kingdom
UK Research and Innovation (UKRI)CC2075, CC2000 United Kingdom
Cancer Research UKCC2075, CC2000 United Kingdom
Wellcome TrustCC2075, CC2000 United Kingdom
Citation
Journal: Nat Commun / Year: 2026
Title: Structure and mechanism of the HECT ligase HECTD3.
Authors: Jessica Huber / Diego Esposito / Sarah Maslen / Dominic O Chambers / J Mark Skehel / Katrin Rittinger /
Abstract: HECT E3 ligases regulate many cellular processes, yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood. HECTD3, a member of ...HECT E3 ligases regulate many cellular processes, yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood. HECTD3, a member of the "other HECT" family, is implicated in the regulation of inflammation, apoptosis, and infection and highly expressed in several cancers. These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains. We present a detailed analysis of the ligase activity of HECTD3, including its ubiquitin linkage preferences, oligomeric state and substrate ubiquitination. Using cryo-EM, we provide the full-length structures of HECTD3 in both apo and ubiquitin-loaded forms, revealing key insights into its domain organisation, including discovery of a distinct fold of the N-terminal region, and mechanistic features. Some of these are shared with other HECT ligases, while others are unique to HECTD3 and contribute to differences in its catalytic mechanisms and functional diversity.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionMay 15, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 28, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Ubiquitin
A: E3 ubiquitin-protein ligase HECTD3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,0183
Polymers105,9612
Non-polymers571
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


Mass: 8519.778 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 (E. coli) / References: UniProt: P0CG48
#2: Protein E3 ubiquitin-protein ligase HECTD3 / HECT domain-containing protein 3 / HECT-type E3 ubiquitin transferase HECTD3


Mass: 97441.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HECTD3 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q5T447, HECT-type E3 ubiquitin transferase
#3: Chemical ChemComp-AYE / prop-2-en-1-amine / ALLYLAMINE


Mass: 57.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7N / Feature type: SUBJECT OF INVESTIGATION
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: HECTD3 ligase conjugated to ubiquitin via activity-based probe ubiquitin propargylamine.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMPhosphateNa-P1
2200 mMSodium ChlorideNaCl1
30.5 mMTCEP1
41.5 %Glycerol1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 41.3 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of real images: 37160
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4RELIONCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3638210
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 342589 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11AF-Q5T4471AlphaFoldin silico model
21UBQ

1ubq

11UBQ2PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00197212
ELECTRON MICROSCOPYf_angle_d0.41869768
ELECTRON MICROSCOPYf_chiral_restr0.03861083
ELECTRON MICROSCOPYf_plane_restr0.00381262
ELECTRON MICROSCOPYf_dihedral_angle_d4.3255977

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