EMDB-47989: E3 ubiquitin ligase HUWE1 homolog Tom1p in closed-conformation wi...

Basic information

Entry

Database: EMDB / ID: EMD-47989

• Title: E3 ubiquitin ligase HUWE1 homolog Tom1p in closed-conformation with internal Acidic Domain deletion.
• Map data: Sharpened map of the C-terminally tagged yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain, in closed-conformation with helical repeat solenoid architecture.

Sample

• Cell: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain (residues 1873-2131).
  • Protein or peptide: E3 ubiquitin-protein ligase TOM1

• Keywords: alpha solenoid / E3 ubiquitin ligase / stress response / cell-cycle / GENE REGULATION

Function / homology

Function:

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

Domain/homology:

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

Component:

E3 ubiquitin-protein ligase TOM1

• Biological species: Saccharomyces cerevisiae (brewer's yeast)
• Method: single particle reconstruction / cryo EM / Resolution: 3.07 Å
• Authors: Madrigal JM

Funding support

United States, 2 items

Organization	Grant number	Country
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

• Citation: Journal: 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 HL / Sdano MA / McCullough L / Connell Z / Formosa T / Hill CP

History

Deposition	Nov 21, 2024	-
Header (metadata) release	Oct 1, 2025	-
Map release	Oct 1, 2025	-
Update	Oct 1, 2025	-
Current status	Oct 1, 2025	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_47989.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Sharpened map of the C-terminally tagged yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain, in closed-conformation with helical repeat solenoid architecture.
• Voxel size: X=Y=Z: 1.12625 Å

Density

Contour Level	By AUTHOR: 0.27
Minimum - Maximum	-3.4387705 - 4.8699923
Average (Standard dev.)	0.0006759767 (±0.0760753)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 360.39996 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_47989_msk_1.map

Additional map: Unsharpened map of the C-terminally tagged yeast E3...

• File: emd_47989_additional_1.map
• Annotation: Unsharpened map of the C-terminally tagged yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain, in closed-conformation with helical repeat solenoid architecture.

Half map: Cryo-EM half-map B of the C-terminally tagged yeast...

• File: emd_47989_half_map_1.map
• Annotation: Cryo-EM half-map B of the C-terminally tagged yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain, in closed-conformation with helical repeat solenoid architecture.

Half map: Cryo-EM half-map A of the C-terminally tagged yeast...

• File: emd_47989_half_map_2.map
• Annotation: Cryo-EM half-map A of the C-terminally tagged yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain, in closed-conformation with helical repeat solenoid architecture.

Sample components

Entire : Yeast E3 ubiquitin ligase Tom1p with an internal truncation at th...

• Entire: Name: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain (residues 1873-2131).

Components

• Cell: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain (residues 1873-2131).
  • Protein or peptide: E3 ubiquitin-protein ligase TOM1

Supramolecule #1: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at th...

• Supramolecule: Name: Yeast E3 ubiquitin ligase Tom1p with an internal truncation at the acidic domain (residues 1873-2131).; type: cell / ID: 1 / Parent: 0 / Macromolecule list: all; Details: C-terminally tagged with Protein A for purification, cleaved with Prescission Protease.
• Source (natural): Organism: Saccharomyces cerevisiae (brewer's yeast)

Macromolecule #1: E3 ubiquitin-protein ligase TOM1

• Macromolecule: Name: E3 ubiquitin-protein ligase TOM1 / type: protein_or_peptide / ID: 1 ; Details: E1873-E2131 deleted in construct Precission Protease tag appended to C-terminus; Number of copies: 1 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
• Source (natural): Organism: Saccharomyces cerevisiae (brewer's yeast)
• Molecular weight: Theoretical: 375.512594 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae (brewer's yeast)

Sequence

String:

MVLFTRCEKA RKEKLAAGYK PLVDYLIDCD TPTFLERIEA IQEWDRSRDD LYVWIPILDR MDGLLLKVAE KYKYKQDPKK ECEVKLVEM EAHDVDYCLK MLKFTRRLLL NTENRFVYSS GDVLMYLLNC PNFTIKLAVM RILAILGERF VIAREKIVAH N IFGDHNLR KKTLKLALSL SSSVMDEDGE HFSLVDLYFD KKKVPQKWRK LRFTHYTSND FKKSSQQKNN INETQTSIKK VT MTTQELC EHSLQQIFDK GMALLPAESW FDFSIKASVA KAFSDDSGEN IDLRNIIIET KLNAIAFVNT IFSPPQVSSK LFE LDPYAF NSLTDLISLS ETKIPKELRT DALFTLECIS LKHVWCSDII RNLGGNISHG LLFQILRYIA KTLREATDEI DEEY NVRFF YLISNLADVK PLHESLFAAG LIPTLLEIVS IRNCPYKRTL ASATHLLETF IDNSETTTEF IENDGFTMLI TSVAN EIDF TLAHPETWQP PKYSVVYYSI SFRELAYIRS LLKLVLKLLS TDSGDRIRNL IDSPILVSLK KILENKLVFG LTLITY TLD VVQKVINSEP TIYPVLVEAG LIPYVIDNFP KLIGPSAELL SLLPDVVSAI CLNPEGLKQV KEKGLINNLF DFLLDAD HA RILTGGDRST EYGTDIDELA RHYPDLKANI VEALCNVIRK MPSTFRNERE FLFTSPKDQK YFFHRKNEEI LTDKEEHE P AYWELLDKGT MLDTFTSVLF GMSLGNGSFS QVPQHLEARD FLAIIFMENP PYEYFTSVAI SNVTEVLQYL DEKYEDYAF MDVMKVLNDQ LENLNDFLNS PNDRSFFLER DGENSVRSCH SKLCRLAAIL NIVTNVYIDL TTLSCKRIMQ IYSYFDKRGF SLIKNLKLL FQKCALEEMY IRQHMPDSVI TETMPLPIVD VSGDGPPLQI YIDDPKKGDQ KGKITSVKTR NTLQMRTILY T LQSNTAIL FRCFLRLSHS RNMDLEHKDL TTEVHIFENV VENVIEMLKA TELEGHLPYI LVLLNFNTFV FTIPKASPNS TE ILQTIPA YIFYQKGGYL LYLHIIRDLF TRMTKIKDLS SLDNINYIDE SNGILTLSCL INALTFYNKS MQTETMENVQ SIG KYYVSI DDDYNIMKAL TVPIKVMALA MILDLDKSDS LFKTQSRNVP YSVFKQLLSM LKNIFTNVNI YTKELYELHW DLIF PPIKK ISLFEQVGIP GDVAANYLTD TGDDLPADNS IGLFSPEQWE KYKKLIGEDK SIYYPQPMQA QYYKGCSSKE LDELR DTFF NDGLPSRIFT VLPFYPKLVN AFAKTLLQIF TKYDEPTEVF AGRILDRILE TDLDDPATLS SLIHLFGIFL NEKYIY QKA SHLMQRFIEY LEKSLKPEHV NTPWFSKALY VYEIILAKSE LPHLEELSKD VLLRYPLLSM AKVFRIPDPM KQKLFDI LI RVSDISNFYS ALATSRILIF YSRDELYANN IARSGILSRL LKVIGSFQKL DKINFLESSF LLLTRRCFET TENVDALI R AEINRSFTAR PLGGGDDAVR ELTTILEEKA HVVMRSPSQF IDVLCETARF HEFDDQGALV DYSLKRFLGE KDKNTQASS TEKSDIYERT GIMHLLLSQL MAASEKDWLS EPANSSDLPE NKKAQLDPSR NPVCAYMIFL LKLLVELVSS YNQCKFEFLT FSRRNTYAE RPRPRTTAIN FFLYRLLDKP VGTDHDKHEA KRREVIGMLA RSVIIGFLAT VQDDRTTKTD VKLADPHMNF I RKFAIEAI IKAIRNATSS SKLLESNHLK LDMWFRIITS MVYVQAPYLR QLLDSNKVEA DQYQLCKLVI DLGLPSVITE AM ASIDLNY PFSKKIFNVA VEALNTISST RNNFSEHFKI EDHDEVEDEV DESDKEEIPD MFKNSALGMY DVEDIEEDDD DDT SLIGDD DAMAFVDSDN GFEVVFSDED DDMGEEDADD ARSDSEENEL SSEMQSSTAD GTDVDYEVDD ADGLIINIDQ PSGD DEEMA DYDANISHSS HSENEDDASM DVIEVYDDEL SSGYDVDLSD YDVDESDWDS GLSSLSISDE DSESSEDEPI NSTRM GDSR RRWLIAEGVE LTDDSQGESE EDDRGVFRGI EHIFSNENEP LFRVHDEMRH RNHHRSINRT HFHSAMSAPS LSLLNR GRR NQSNLINPLG PTGLEQVEND ISDQVTVAGS GSRPRSHHLH FSEVLVSGSF FDEPVLDGII LKSTVSRWKD IFDMFYD SK TYANCIIPTV INRLYKVSLA LQKDLENKRE QEKLKNKNLL FNEAKVESHN SSDAISVEQD DIQESNVTHD DHEPVYVT I QGSEVDIGGT DIDPEFMNAL PDDIRADVFA QHVRERRAEA RLNSDHNVHS REIDSDFLEA IPEDIREGIL DTEAEEQRM FGRIGSSADV IRADDDVSNN DEEVENGLDH GNSNDRNNAD PEKKKPARIY FAPLIDRAGI ASLMKSVFIS KPYIQREIYH ELFYRLCSS KQNRNDLMNT FLFILSEGII DQHSLEKVYN IISSRAMGHA KTTTVRQLPS DCTPLTVANQ TIEILQSLID A DSRLKYFL IAEHDNLIVN KANNKSRKEA LPDKKLRWPL WHLFSLLDRK LITDESVLMD LLTRILQVCT KTLAVLSTSS NG KENLSKK FHLPSFDEDD LMKILSIIML DSCTTRVFQQ TLNIIYNLSK LQGCMSIFTK HLVSLAISIM SKLKSALDGL SRE VGTITT GMEINSELLQ KFTLPSSDQA KLLKILTTVD FLYTHKRKEE ERNVKDLQSL YDKMNGGPVW SSLSECLSQF EKSQ AINTS ATILLPLIES LMVVCRRSDL SQNRNTAVKY EDAKLLDFSK TRVENLFFPF TDAHKKLLNQ MIRSNPKLMS GPFAL LVKN PKVLDFDNKR YFFNAKLKSD NQERPKLPIT VRREQVFLDS YRALFFKTND EIKNSKLEIT FKGESGVDAG GVTREW YQV LSRQMFNPDY ALFLPVPSDK TTFHPNRTSG INPEHLSFFK FIGMIIGKAI RDQCFLDCHF SREVYKNILG RPVSLKD ME SLDPDYYKSL VWILENDITD IIEETFSVET DDYGEHKVIN LIEGGKDIIV TEANKQDYVK KVVEYKLQTS VKEQMDNF L VGFYALISKD LITIFDEQEL ELLISGLPDI DVDDWKNNTT YVNYTATCKE VSYFWRAVRS FDAEERAKLL QFVTGTSKV PLNGFKELSG VNGVCKFSIH RDFGSSERLP SSHTCFNQLN LPPYESYETL RGSLLLAINE GHEGFGLALE VLFQGP

UniProtKB: E3 ubiquitin-protein ligase TOM1

Experimental details

Structure determination

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

Sample preparation

• Concentration: 1 mg/mL
• Buffer: pH: 7
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I; 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..

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Number real images: 7236 / Average electron dose: 47.0 e/Ų; 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.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Software - Name: cryoSPARC / Type: NONE
• Startup model: Type of model: NONE; Details: Initial blob auto-picking, 2D classification, ab initio reconstructions and heterogeneous refinements from the non-detergent sample led to 3 refined volumes. Templates were low-pass filtered to 17 A, creating 25 equally spaced templates each for a total of 75 templates. These templates were used to template-pick from both non-detergent and detergent sample datasets.
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Non-uniform refinement; 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.; Number images used: 462711
• Initial angle assignment: Type: RANDOM ASSIGNMENT
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final 3D classification: Software - Name: cryoSPARC