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- EMDB-47035: Cryo-EM map of full-length Tom1 (S. cerevisiae), HECT focus map -

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Basic information

Entry
Database: EMDB / ID: EMD-47035
TitleCryo-EM map of full-length Tom1 (S. cerevisiae), HECT focus map
Map dataconsensus, Tom1 BS3 crosslink HECT domain focus map
Sample
  • Complex: Full length structure, S. cerevisiae Tom1
    • Protein or peptide: E3 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWarner KM / Hunkeler M / Fischer ES
Funding support United States, 2 items
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 17, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47035.png

Downloads & links

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Map

FileDownload / File: emd_47035.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationconsensus, Tom1 BS3 crosslink HECT domain focus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.37915686 - 0.64909583
Average (Standard dev.)0.00030186787 (±0.009499375)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47035_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_47035_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer map, Tom1 BS3 crosslink HECT domain focus map

Fileemd_47035_additional_1.map
AnnotationDeepEMhancer map, Tom1 BS3 crosslink HECT domain focus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A, Tom1 BS3 crosslink HECT domain focus map

Fileemd_47035_half_map_1.map
Annotationhalf map A, Tom1 BS3 crosslink HECT domain focus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B, Tom1 BS3 crosslink HECT domain focus map

Fileemd_47035_half_map_2.map
Annotationhalf map B, Tom1 BS3 crosslink HECT domain focus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Full length structure, S. cerevisiae Tom1

EntireName: Full length structure, S. cerevisiae Tom1
Components
  • Complex: Full length structure, S. cerevisiae Tom1
    • Protein or peptide: E3 ubiquitin-protein ligase Tom1

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Supramolecule #1: Full length structure, S. cerevisiae Tom1

SupramoleculeName: Full length structure, S. cerevisiae Tom1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Tom1 closed-ring conformation, stabilized
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 376 KDa

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Macromolecule #1: E3 ubiquitin-protein ligase Tom1

MacromoleculeName: E3 ubiquitin-protein ligase Tom1 / type: protein_or_peptide / ID: 1 / Details: StrepIItag-TEV-Tom1 fusion / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDWSHPQFEK SAVDENLYFQ GGGRMVLFTR CEKARKEKLA AGYKPLVDYL IDCDTPTFLE RIEAIQEWDR SRDDLYVWIP ILDRMDGLLL KVAEKYKYKQ DPKKECEVKL VEMEAHDVDY CLKMLKFTRR LLLNTENRFV YSSGDVLMYL LNCPNFTIKL AVMRILAILG ...String:
MDWSHPQFEK SAVDENLYFQ GGGRMVLFTR CEKARKEKLA AGYKPLVDYL IDCDTPTFLE RIEAIQEWDR SRDDLYVWIP ILDRMDGLLL KVAEKYKYKQ DPKKECEVKL VEMEAHDVDY CLKMLKFTRR LLLNTENRFV YSSGDVLMYL LNCPNFTIKL AVMRILAILG ERFVIAREKI VAHNIFGDHN LRKKTLKLAL SLSSSVMDED GEHFSLVDLY FDKKKVPQKW RKLRFTHYTS NDFKKSSQQK NNINETQTSI KKVTMTTQEL CEHSLQQIFD KGMALLPAES WFDFSIKASV AKAFSDDSGE NIDLRNIIIE TKLNAIAFVN TIFSPPQVSS KLFELDPYAF NSLTDLISLS ETKIPKELRT DALFTLECIS LKHVWCSDII RNLGGNISHG LLFQILRYIA KTLREATDEI DEEYNVRFFY LISNLADVKP LHESLFAAGL IPTLLEIVSI RNCPYKRTLA SATHLLETFI DNSETTTEFI ENDGFTMLIT SVANEIDFTL AHPETWQPPK YSVVYYSISF RELAYIRSLL KLVLKLLSTD SGDRIRNLID SPILVSLKKI LENKLVFGLT LITYTLDVVQ KVINSEPTIY PVLVEAGLIP YVIDNFPKLI GPSAELLSLL PDVVSAICLN PEGLKQVKEK GLINNLFDFL LDADHARILT GGDRSTEYGT DIDELARHYP DLKANIVEAL CNVIRKMPST FRNEREFLFT SPKDQKYFFH RKNEEILTDK EEHEPAYWEL LDKGTMLDTF TSVLFGMSLG NGSFSQVPQH LEARDFLAII FMENPPYEYF TSVAISNVTE VLQYLDEKYE DYAFMDVMKV LNDQLENLND FLNSPNDRSF FLERDGENSV RSCHSKLCRL AAILNIVTNV YIDLTTLSCK RIMQIYSYFD KRGFSLIKNL KLLFQKCALE EMYIRQHMPD SVITETMPLP IVDVSGDGPP LQIYIDDPKK GDQKGKITSV KTRNTLQMRT ILYTLQSNTA ILFRCFLRLS HSRNMDLEHK DLTTEVHIFE NVVENVIEML KATELEGHLP YILVLLNFNT FVFTIPKASP NSTEILQTIP AYIFYQKGGY LLYLHIIRDL FTRMTKIKDL SSLDNINYID ESNGILTLSC LINALTFYNK SMQTETMENV QSIGKYYVSI DDDYNIMKAL TVPIKVMALA MILDLDKSDS LFKTQSRNVP YSVFKQLLSM LKNIFTNVNI YTKELYELHW DLIFPPIKKI SLFEQVGIPG DVAANYLTDT GDDLPADNSI GLFSPEQWEK YKKLIGEDKS IYYPQPMQAQ YYKGCSSKEL DELRDTFFND GLPSRIFTVL PFYPKLVNAF AKTLLQIFTK YDEPTEVFAG RILDRILETD LDDPATLSSL IHLFGIFLNE KYIYQKASHL MQRFIEYLEK SLKPEHVNTP WFSKALYVYE IILAKSELPH LEELSKDVLL RYPLLSMAKV FRIPDPMKQK LFDILIRVSD ISNFYSALAT SRILIFYSRD ELYANNIARS GILSRLLKVI GSFQKLDKIN FLESSFLLLT RRCFETTENV DALIRAEINR SFTARPLGGG DDAVRELTTI LEEKAHVVMR SPSQFIDVLC ETARFHEFDD QGALVDYSLK RFLGEKDKNT QASSTEKSDI YERTGIMHLL LSQLMAASEK DWLSEPANSS DLPENKKAQL DPSRNPVCAY MIFLLKLLVE LVSSYNQCKF EFLTFSRRNT YAERPRPRTT AINFFLYRLL DKPVGTDHDK HEAKRREVIG MLARSVIIGF LATVQDDRTT KTDVKLADPH MNFIRKFAIE AIIKAIRNAT SSSKLLESNH LKLDMWFRII TSMVYVQAPY LRQLLDSNKV EADQYQLCKL VIDLGLPSVI TEAMASIDLN YPFSKKIFNV AVEALNTISS TRNNFSEHFK IEDHDEVEDE VDESDKEEIP DMFKNSALGM YDVEDIEEDD DDDTSLIGDD DAMAFVDSDN GFEVVFSDED DDMGEEDADD ARSDSEENEL SSEMQSSTAD GTDVDYEVDD ADGLIINIDQ PSGDDEEMAD YDANISHSSH SENEDDASMD VIEVYDDELS SGYDVDLSDY DVDESDWDSG LSSLSISDED SESSEDEPIN STRMGDSRRR WLIAEGVELT DDSQGESEED DRGVFRGIEH IFSNENEPLF RVHDEMRHRN HHRSINRTHF HSAMSAPSLS LLNRGRRNQS NLINPLGPTG LEQVENDISD QVTVAGSGSR PRSHHLHFSE VLVSGSFFDE PVLDGIILKS TVSRWKDIFD MFYDSKTYAN CIIPTVINRL YKVSLALQKD LENKREQEKL KNKNLLFNEA KVESHNSSDA ISVEQDDIQE SNVTHDDHEP VYVTIQGSEV DIGGTDIDPE FMNALPDDIR ADVFAQHVRE RRAEARLNSD HNVHSREIDS DFLEAIPEDI REGILDTEAE EQRMFGRIGS SADVIRADDD VSNNDEEVEN GLDHGNSNDR NNADPEKKKP ARIYFAPLID RAGIASLMKS VFISKPYIQR EIYHELFYRL CSSKQNRNDL MNTFLFILSE GIIDQHSLEK VYNIISSRAM GHAKTTTVRQ LPSDCTPLTV ANQTIEILQS LIDADSRLKY FLIAEHDNLI VNKANNKSRK EALPDKKLRW PLWHLFSLLD RKLITDESVL MDLLTRILQV CTKTLAVLST SSNGKENLSK KFHLPSFDED DLMKILSIIM LDSCTTRVFQ QTLNIIYNLS KLQGCMSIFT KHLVSLAISI MSKLKSALDG LSREVGTITT GMEINSELLQ KFTLPSSDQA KLLKILTTVD FLYTHKRKEE ERNVKDLQSL YDKMNGGPVW SSLSECLSQF EKSQAINTSA TILLPLIESL MVVCRRSDLS QNRNTAVKYE DAKLLDFSKT RVENLFFPFT DAHKKLLNQM IRSNPKLMSG PFALLVKNPK VLDFDNKRYF FNAKLKSDNQ ERPKLPITVR REQVFLDSYR ALFFKTNDEI KNSKLEITFK GESGVDAGGV TREWYQVLSR QMFNPDYALF LPVPSDKTTF HPNRTSGINP EHLSFFKFIG MIIGKAIRDQ CFLDCHFSRE VYKNILGRPV SLKDMESLDP DYYKSLVWIL ENDITDIIEE TFSVETDDYG EHKVINLIEG GKDIIVTEAN KQDYVKKVVE YKLQTSVKEQ MDNFLVGFYA LISKDLITIF DEQELELLIS GLPDIDVDDW KNNTTYVNYT ATCKEVSYFW RAVRSFDAEE RAKLLQFVTG TSKVPLNGFK ELSGVNGVCK FSIHRDFGSS ERLPSSHTCF NQLNLPPYES YETLRGSLLL AINEGHEGFG LA

UniProtKB: E3 ubiquitin-protein ligase TOM1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.8 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
30.0 mMHEPESHEPES
2.0 mMTCEPtris(2-carboxyethyl)phosphine
0.3 mMCHAPSOCHAPSO

Details: 30 mM HEPES pH 7.2, 200 mM NaCl, 2 mM TCEP, 0.3 mM CHAPSO
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa / Details: 60 seconds, hold time 10 seconds, Current 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA EM GP
Details: CHAPSO detergent added to final conc. of 0.3 mM. Sample applied twice..

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11583 / Average exposure time: 2.8 sec. / Average electron dose: 53.687 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4216890
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 174094
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3280


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model

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