EMDB-53804: Cryo-EM structure of the E3 ligase HECTD3 conjugated to ubiquitin

Basic information

Entry

Database: EMDB / ID: EMD-53804

• Title: Cryo-EM structure of the E3 ligase HECTD3 conjugated to ubiquitin
• Map data: Main map

Sample

• Complex: HECTD3 ligase conjugated to ubiquitin via activity-based probe ubiquitin propargylamine.
  • Protein or peptide: Ubiquitin
  • Protein or peptide: E3 ubiquitin-protein ligase HECTD3
• Ligand: prop-2-en-1-amine

• Keywords: E3-ligase / ubiquitin / ubiquitination / HECT-ligase / LIGASE

Function / homology

Function:

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 / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Prevention of phagosomal-lysosomal fusion / Endosomal Sorting Complex Required For Transport (ESCRT) / Membrane binding and targetting of GAG proteins / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Constitutive Signaling by NOTCH1 HD Domain Mutants / 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 / APC-Cdc20 mediated degradation of Nek2A / Regulation of FZD by ubiquitination / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / NF-kB is activated and signals survival / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Pexophagy / Downregulation of ERBB2:ERBB3 signaling / Regulation of innate immune responses to cytosolic DNA / 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 / Translesion synthesis by REV1 / TICAM1, RIP1-mediated IKK complex recruitment / Regulation of BACH1 activity / Translesion synthesis by POLK / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / InlB-mediated entry of Listeria monocytogenes into host cell / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / Josephin domain DUBs / Gap-filling DNA repair synthesis and ligation in GG-NER / IKK complex recruitment mediated by RIP1 / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / Regulation of activated PAK-2p34 by proteasome mediated degradation / TCF dependent signaling in response to WNT / Regulation of NF-kappa B signaling / activated TAK1 mediates p38 MAPK activation / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / NOTCH3 Activation and Transmission of Signal to the Nucleus / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Regulation of signaling by CBL / Negative regulators of DDX58/IFIH1 signaling / Asymmetric localization of PCP proteins / Fanconi Anemia Pathway / Ubiquitin-dependent degradation of Cyclin D / Negative regulation of FGFR3 signaling / Peroxisomal protein import / Deactivation of the beta-catenin transactivating complex / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Stabilization of p53 / Negative regulation of FGFR2 signaling / Negative regulation of FGFR4 signaling / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Assembly of the pre-replicative complex / Negative regulation of FGFR1 signaling / Termination of translesion DNA synthesis / Vpu mediated degradation of CD4 / EGFR downregulation / Regulation of TNFR1 signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Assembly Of The HIV Virion / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of DVL / Degradation of AXIN / Degradation of CRY and PER proteins / Spry regulation of FGF signaling / Late endosomal microautophagy / Hh mutants are degraded by ERAD / Activation of NF-kappaB in B cells / Iron uptake and transport

Domain/homology:

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

Component:

Polyubiquitin-C / E3 ubiquitin-protein ligase HECTD3

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.04 Å
• Authors: Esposito D / Huber J / Maslen S / Rittinger K

Funding support

United Kingdom, 4 items

Organization	Grant number	Country
The Francis Crick Institute	CC2075, CC2000	United Kingdom
UK Research and Innovation (UKRI)	CC2075, CC2000	United Kingdom
Cancer Research UK	CC2075, CC2000	United Kingdom
Wellcome Trust	CC2075, CC2000	United Kingdom

• Citation: 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 / 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 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

Deposition	May 15, 2025	-
Header (metadata) release	Jan 28, 2026	-
Map release	Jan 28, 2026	-
Update	Apr 8, 2026	-
Current status	Apr 8, 2026	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_53804.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Main map
• Voxel size: X=Y=Z: 0.95 Å

Density

Contour Level	By AUTHOR: 0.142
Minimum - Maximum	-0.31553128 - 0.88626593
Average (Standard dev.)	0.00034200453 (±0.017626977)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 296 296 296 Spacing 296 296 296
Cell	A=B=C: 281.19998 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Local filtered map used in phenix.refine

• File: emd_53804_additional_1.map
• Annotation: Local filtered map used in phenix.refine

Additional map: EMReady sharpened and enhanced map.

• File: emd_53804_additional_2.map
• Annotation: EMReady sharpened and enhanced map.

Half map: Half-map

• File: emd_53804_half_map_1.map
• Annotation: Half-map

Half map: Half-map

• File: emd_53804_half_map_2.map
• Annotation: Half-map

Sample components

Entire : HECTD3 ligase conjugated to ubiquitin via activity-based probe ub...

• Entire: Name: HECTD3 ligase conjugated to ubiquitin via activity-based probe ubiquitin propargylamine.

Components

• Complex: HECTD3 ligase conjugated to ubiquitin via activity-based probe ubiquitin propargylamine.
  • Protein or peptide: Ubiquitin
  • Protein or peptide: E3 ubiquitin-protein ligase HECTD3
• Ligand: prop-2-en-1-amine

Supramolecule #1: HECTD3 ligase conjugated to ubiquitin via activity-based probe ub...

• Supramolecule: Name: HECTD3 ligase conjugated to ubiquitin via activity-based probe ubiquitin propargylamine.; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Ubiquitin

• Macromolecule: Name: Ubiquitin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 8.519778 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG

UniProtKB: Polyubiquitin-C

Macromolecule #2: E3 ubiquitin-protein ligase HECTD3

• Macromolecule: Name: E3 ubiquitin-protein ligase HECTD3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 97.441055 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

GPGMAGPGPG AVLESPRQLL GRVRFLAEAA RSLRAGRPLP AALAFVPREV LYKLYKDPAG PSRVLLPVWE AEGLGLRVGA AGPAPGTGS GPLRAARDSI ELRRGACVRT TGEELCNGHG LWVKLTKEQL AEHLGDCGLQ EGWLLVCRPA EGGARLVPID T PNHLQRQQ QLFGVDYRPV LRWEQVVDLT YSHRLGSRPQ PAEAYAEAVQ RLLYVPPTWT YECDEDLIHF LYDHLGKEDE NL GSVKQYV ESIDVSSYTE EFNVSCLTDS NADTYWESDG SQCQHWVRLT MKKGTIVKKL LLTVDTTDDN FMPKRVVVYG GEG DNLKKL SDVSIDETLI GDVCVLEDMT VHLPIIEIRI VECRDDGIDV RLRGVKIKSS RQRELGLNAD LFQPTSLVRY PRLE GTDPE VLYRRAVLLQ RFIKILDSVL HHLVPAWDHT LGTFSEIKQV KQFLLLSRQR PGLVAQCLRD SESSKPSFMP RLYIN RRLA MEHRACPSRD PACKNAVFTQ VYEGLKPSDK YEKPLDYRWP MRYDQWWECK FIAEGIIDQG GGFRDSLADM SEELCP SSA DTPVPLPFFV RTANQGNGTG EARDMYVPNP SCRDFAKYEW IGQLMGAALR GKEFLVLALP GFVWKQLSGE EVSWSKD FP AVDSVLVKLL EVMEGMDKET FEFKFGKELT FTTVLSDQQV VELIPGGAGI VVGYGDRSRF IQLVQKARLE ESKEQVAA M QAGLLKVVPQ AVLDLLTWQE LEKKVCGDPE VTVDALRKLT RFEDFEPSDS RVQYFWEALN NFTNEDRSRF LRFVTGRSR LPARIYIYPD KLGYETTDAL PESSTCSSTL FLPHYASAKV CEEKLRYAAY NCVAIDTDMS PWEE

UniProtKB: E3 ubiquitin-protein ligase HECTD3

Macromolecule #3: prop-2-en-1-amine

• Macromolecule: Name: prop-2-en-1-amine / type: ligand / ID: 3 / Number of copies: 1 / Formula: AYE
• Molecular weight: Theoretical: 57.094 Da

Chemical component information

ChemComp-AYE:
prop-2-en-1-amine

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.5 mg/mL

Buffer

pH: 7
Component:

Concentration	Formula	Name
50.0 mM	Na-P	Phosphate
200.0 mM	NaCl	Sodium Chloride
0.5 mM		TCEP
1.5 %		Glycerol

• Grid: Model: C-flat / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: TFS KRIOS
• Specialist optics: Energy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
• Image recording: Film or detector model: TFS FALCON 4i (4k x 4k) / Number real images: 37160 / Average electron dose: 41.3 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 3638210
• CTF correction: Software - Name: RELION / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: NONE
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 342589
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

Atomic model buiding 1

Initial model

PDB ID	Chain
t447	source_name: AlphaFold, initial_model_type: in silico model
1ubq	source_name: PDB, initial_model_type: experimental model

• Refinement: Space: REAL / Protocol: BACKBONE TRACE

Output model

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