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- PDB-9egk: E3 ubiquitin ligase HUWE1 homolog Tom1p in closed-conformation wi... -

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Basic information

Entry
Database: PDB / ID: 9egk
TitleE3 ubiquitin ligase HUWE1 homolog Tom1p in closed-conformation with internal Acidic Domain deletion
ComponentsE3 ubiquitin-protein ligase TOM1
KeywordsGENE REGULATION / alpha solenoid / E3 ubiquitin ligase / stress response / cell-cycle
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 / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of cell size / 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 / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of cell size / 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: 3.07 Å
AuthorsMadrigal, J.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54AI170856 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM064649 United States
CitationJournal: Elife / Year: 2024
Title: Tom1p ubiquitin ligase structure, interaction with Spt6p, and function in maintaining normal transcript levels and the stability of chromatin in promoters
Authors: Madrigal, J. / Schubert, H.L. / Sdano, M.A. / McCullough, L. / Connell, Z. / Formosa, T. / Hill, C.P.
History
DepositionNov 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase TOM1


Theoretical massNumber of molelcules
Total (without water)375,5131
Polymers375,5131
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: 375512.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: E1873-E2131 deleted in construct Precission Protease tag appended to C-terminus
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TOM1, SSR2, YDR457W, D8035.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): 9750
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: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain (residues 1873-2131).
Type: CELL
Details: C-terminally tagged with Protein A for purification, cleaved with Prescission Protease.
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %
Details: Specimens were prepared on Quantifoil R 2/2 Cu300 grids after glow discharge for 1 minute at 25 mA. The detergent sample was concentrated to 8.1 mg/mL in 0.01% CHAPS and 0.05% NP-40. 2.5 uL ...Details: Specimens were prepared on Quantifoil R 2/2 Cu300 grids after glow discharge for 1 minute at 25 mA. The detergent sample was concentrated to 8.1 mg/mL in 0.01% CHAPS and 0.05% NP-40. 2.5 uL of sample was applied to grids and blotted for 2.5 seconds before vitrification by plunging into liquid ethane. For samples without detergent, protein was concentrated to 0.44 mg/mL and blotted for 1.5 seconds before vitrification.

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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: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7236
Details: A total of 2,865 movies were recorded from grids without detergent at 81,000x magnification on a 300 kV Titan Krios G3 electron microscope with a K3 direct detector (nominal resolution after ...Details: A total of 2,865 movies were recorded from grids without detergent at 81,000x magnification on a 300 kV Titan Krios G3 electron microscope with a K3 direct detector (nominal resolution after 2x binning is 1.06A/px). Electron exposure was 47 (e-/A2) over a total of 40 frames. A total of 4,371 movies were recorded from with-detergent grids using the same electron microscope and collection parameters.

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selection
4cryoSPARCCTF correction
8PHENIXmodel refinement
12cryoSPARCclassification
13cryoSPARC3D reconstructionNon-uniform refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462711
Details: The separately processed dataset #1 and #2 particles were combined for a total of 531,549 starting particles for further processing and refinement. A round of 2D classification resulted in ...Details: The separately processed dataset #1 and #2 particles were combined for a total of 531,549 starting particles for further processing and refinement. A round of 2D classification resulted in selection of 477,889 particles, which were Fourier cropped from a box size of 680 to 320 pixels for further processing. Two-component 3DVA was performed after map refinement of the combined dataset with a filter resolution of 4 A. 3DVA display was subsequently run to give 8 intermediates along the first principal component at a filter resolution of 5 A. The top 4 intermediates with the highest number of particles were combined to yield 462,711 and refined using non-uniform refinement to an estimated overall resolution of 3.07 A.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422727
ELECTRON MICROSCOPYf_angle_d0.60130773
ELECTRON MICROSCOPYf_dihedral_angle_d4.9453007
ELECTRON MICROSCOPYf_chiral_restr0.0553557
ELECTRON MICROSCOPYf_plane_restr0.0053900

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