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- PDB-9dns: Cryo-EM structure of Tom1-UBE2D2-ubiquitin complex -

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Basic information

Entry
Database: PDB / ID: 9dns
TitleCryo-EM structure of Tom1-UBE2D2-ubiquitin complex
Components
  • E3 ubiquitin-protein ligase TOM1
  • Ubiquitin
  • Ubiquitin-conjugating enzyme E2 D2
Keywordstransferase/ligase / transferase / ubiquitin / ubiquitylation / ligase / transferase-ligase complex
Function / homology
Function and homology information


endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / (E3-independent) E2 ubiquitin-conjugating enzyme / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / E2 ubiquitin-conjugating enzyme / regulation of cell size / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nucleus organization / ubiquitin conjugating enzyme activity ...endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / (E3-independent) E2 ubiquitin-conjugating enzyme / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / E2 ubiquitin-conjugating enzyme / regulation of cell size / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nucleus organization / ubiquitin conjugating enzyme activity / Antigen processing: Ubiquitination & Proteasome degradation / mRNA transport / protein K48-linked ubiquitination / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / protein autoubiquitination / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Neutrophil degranulation / 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 / Regulation of innate immune responses to cytosolic DNA / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / 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 / Translesion synthesis by POLK / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / InlB-mediated entry of Listeria monocytogenes into host cell / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Translesion synthesis by POLI / Regulation of activated PAK-2p34 by proteasome mediated degradation / IKK complex recruitment mediated by RIP1 / 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 / 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 / Ubiquitin-dependent degradation of Cyclin D / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / activated TAK1 mediates p38 MAPK activation / Negative regulators of DDX58/IFIH1 signaling / TNFR2 non-canonical NF-kB pathway / AUF1 (hnRNP D0) binds and destabilizes mRNA / Regulation of signaling by CBL / NOTCH3 Activation and Transmission of Signal to the Nucleus / Vpu mediated degradation of CD4 / Assembly of the pre-replicative complex / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Degradation of DVL / Deactivation of the beta-catenin transactivating complex / Negative regulation of FGFR3 signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Dectin-1 mediated noncanonical NF-kB signaling / Fanconi Anemia Pathway / Peroxisomal protein import / Degradation of AXIN / Regulation of TNFR1 signaling
Similarity search - Function
E3 ubiquitin ligase, domain of unknown function DUF908 / E3 ubiquitin ligase, domain of unknown function DUF913 / Domain of Unknown Function (DUF908) / Domain of Unknown Function (DUF913) / HUWE1/Rev1, ubiquitin binding region / Ubiquitin binding region / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) ...E3 ubiquitin ligase, domain of unknown function DUF908 / E3 ubiquitin ligase, domain of unknown function DUF913 / Domain of Unknown Function (DUF908) / Domain of Unknown Function (DUF913) / HUWE1/Rev1, ubiquitin binding region / Ubiquitin binding region / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Armadillo-like helical / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Armadillo-type fold / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Polyubiquitin-C / Ubiquitin-conjugating enzyme E2 D2 / E3 ubiquitin-protein ligase TOM1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsWarner, K.M. / Hunkeler, M. / Baek, K. / Roy Burman, S.S. / Fischer, E.S.
Funding support United States, 2items
OrganizationGrant numberCountry
The G. Harold and Leila Y. Mathers Foundation64395403 United States
Damon Runyon Cancer Research FoundationDRG-2514-24 United States
CitationJournal: Cell Rep / Year: 2025
Title: Structural ubiquitin contributes to K48 linkage specificity of the HECT ligase Tom1.
Authors: Katrina Warner / Moritz Hunkeler / Kheewoong Baek / Anna Schmoker / Shourya S Roy Burman / Daan Overwijn / Cyrus Jin / Katherine A Donovan / Eric S Fischer /
Abstract: Homologous to E6AP C terminus (HECT) ubiquitin ligases play key roles in essential pathways such as DNA repair, cell cycle control, or protein quality control. Tom1 is one of five HECT ubiquitin E3 ...Homologous to E6AP C terminus (HECT) ubiquitin ligases play key roles in essential pathways such as DNA repair, cell cycle control, or protein quality control. Tom1 is one of five HECT ubiquitin E3 ligases in budding yeast S. cerevisiae and is prototypical for a ligase with pleiotropic functions such as ubiquitin chain amplification, orphan quality control, and DNA damage response. Structures of full-length HECT ligases, including the Tom1 ortholog HUWE1, have been reported, but how domains beyond the conserved catalytic module contribute to catalysis remains largely elusive. Here, through cryoelectron microscopy (cryo-EM) snapshots of Tom1 during an active ubiquitination cycle, we demonstrate that the extended domain architecture directly contributes to activity. We identify a Tom1-ubiquitin architecture during ubiquitination involving a non-canonical ubiquitin-binding site in the solenoid shape of Tom1. We demonstrate that this ubiquitin-binding site coordinates a structural ubiquitin contributing to the fidelity of K48 poly-ubiquitin chain assembly.
History
DepositionSep 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase TOM1
D: Ubiquitin-conjugating enzyme E2 D2
E: Ubiquitin-conjugating enzyme E2 D2
U: Ubiquitin
V: Ubiquitin
W: Ubiquitin
X: Ubiquitin


Theoretical massNumber of molelcules
Total (without water)445,5197
Polymers445,5197
Non-polymers00
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 E3 ubiquitin-protein ligase TOM1 / HECT-type E3 ubiquitin transferase TOM1 / Suppressor of snRNA protein 2 / Temperature-dependent ...HECT-type E3 ubiquitin transferase TOM1 / Suppressor of snRNA protein 2 / Temperature-dependent organization in mitotic nucleus protein 1


Mass: 377412.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: StrepIItag-TEV-Tom1 fusion
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TOM1, SSR2, YDR457W, D8035.1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q03280, HECT-type E3 ubiquitin transferase
#2: Protein Ubiquitin-conjugating enzyme E2 D2 / (E3-independent) E2 ubiquitin-conjugating enzyme D2 / E2 ubiquitin-conjugating enzyme D2 / ...(E3-independent) E2 ubiquitin-conjugating enzyme D2 / E2 ubiquitin-conjugating enzyme D2 / Ubiquitin carrier protein D2 / Ubiquitin-conjugating enzyme E2(17)KB 2 / Ubiquitin-conjugating enzyme E2-17 kDa 2 / Ubiquitin-protein ligase D2 / p53-regulated ubiquitin-conjugating enzyme 1


Mass: 16899.357 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: TEV cleavage product GST-TEV-UBE2D2 / Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2D2, PUBC1, UBC4, UBC5B, UBCH4, UBCH5B / Production host: Escherichia coli (E. coli)
References: UniProt: P62837, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme
#3: Protein
Ubiquitin


Mass: 8576.831 Da / Num. of mol.: 4
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
Has protein modificationN

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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: Ternary complex of E3 ubiquitin ligase Tom1 with E2 ubiquitin conjugating enzyme UBE2D2 and ubiquitin
Type: COMPLEX
Details: Complex identified by plunge-freezing Tom1 in active ubiquitylation cascade
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.444 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.2
Details: 30 mM HEPES pH 7.2, 200 mM NaCl, 0.5 mM TCEP, 0.2 mM CHAPSO
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
230 mMHEPESHEPES1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
40.2 mMCHAPSOCHAPSO1
SpecimenConc.: 1.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was prepared by pre-forming Tom1:H2B complex. In tandem, UBE2D2/UBA1/ubiquitin were incubated in reaction buffer containing Mg-ATP for twenty minutes at room temperature, then buffer ...Details: Sample was prepared by pre-forming Tom1:H2B complex. In tandem, UBE2D2/UBA1/ubiquitin were incubated in reaction buffer containing Mg-ATP for twenty minutes at room temperature, then buffer exchanged. The enzymatic cascade was initiated by mixing both samples and quenched by plunge freezing. Final sample concentrations were: 5 uM Tom1, 10 uM histone H2B, 0.25 uM UBA1, 44 uM UBE2D2, 75 uM ubiquitin, 0.2 mM CHAPSO.
Specimen supportDetails: 60 seconds / hold time: 10 seconds / Current: 15 mA
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K
Details: CHAPSO detergent added to final conc. of 0.2 mM. Sample applied twice.

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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: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 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: 2.896 sec. / Electron dose: 56.13 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18959
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2SerialEM4.1bimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fitting
10cryoSPARC4.4.1initial Euler assignment
11cryoSPARC4.4.1final Euler assignment
12cryoSPARC4.4.1classification
13cryoSPARC4.4.13D reconstruction
14PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11388836
Details: Initial 2D classes assisted template picking in cryoSPARC Live.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 246341 / Symmetry type: POINT
Atomic model buildingB value: 89.6 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient / Details: Phenix real-space refinement
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeSource nameTypeChain-IDInitial refinement model-ID
1Q03280AlphaFoldin silico model
24V3L

4v3l

A4V3LPDBexperimental modelA2
34V3L

4v3l

B4V3LPDBexperimental modelB2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226186
ELECTRON MICROSCOPYf_angle_d0.44535440
ELECTRON MICROSCOPYf_dihedral_angle_d4.3173462
ELECTRON MICROSCOPYf_chiral_restr0.0364079
ELECTRON MICROSCOPYf_plane_restr0.0034486

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