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- PDB-9dnt: Cryo-EM structure of Tom1 (S. cerevisiae) -

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Basic information

Entry
Database: PDB / ID: 9dnt
TitleCryo-EM structure of Tom1 (S. cerevisiae)
ComponentsE3 ubiquitin-protein ligase TOM1
KeywordsTRANSFERASE / ubiquitin / ubiquitylation 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) / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / 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 / Antigen processing: Ubiquitination & Proteasome degradation / mRNA transport / 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) ...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) / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / 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 / Antigen processing: Ubiquitination & Proteasome degradation / mRNA transport / 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) / Neutrophil degranulation / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein ubiquitination / nucleolus / nucleus / cytoplasm
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 / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
E3 ubiquitin-protein ligase TOM1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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


Theoretical massNumber of molelcules
Total (without water)377,4131
Polymers377,4131
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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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
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
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: Full length structure, S. cerevisiae Tom1 / Type: COMPLEX / Details: Tom1 closed-ring conformation, stabilized / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.376 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, 2 mM TCEP, 0.3 mM CHAPSO
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
230 mMHEPESHEPES1
32 mMtris(2-carboxyethyl)phosphineTCEP1
40.3 mMCHAPSOCHAPSO1
SpecimenConc.: 4.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 283.15 K
Details: CHAPSO detergent added to final conc. of 0.3 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: 1100 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: 2.8 sec. / Electron dose: 53.687 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11583
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2SerialEM4.1bimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fitting
10PHENIX1.19.2-4158model refinement
11cryoSPARC4.4.1initial Euler assignment
12cryoSPARC4.4.1final Euler assignment
13cryoSPARC4.4.1classification
14cryoSPARC4.4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4216890
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 451216 / Symmetry type: POINT
Atomic model buildingB value: 121
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320476
ELECTRON MICROSCOPYf_angle_d0.42327680
ELECTRON MICROSCOPYf_dihedral_angle_d4.8462706
ELECTRON MICROSCOPYf_chiral_restr0.0343215
ELECTRON MICROSCOPYf_plane_restr0.0033461

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