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- EMDB-47049: Tom1 ubiquitylation cascade, closed-ring conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-47049
TitleTom1 ubiquitylation cascade, closed-ring conformation
Map dataTom1 ubiquitylation cascade, closed-ring, consensus
Sample
  • Complex: Tom1 ubiquitylation cascade, closed-ring conformation
    • 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: 3.7 Å
AuthorsWarner KM / Hunkeler M / Baek K / 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 18, 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_47049.png

Downloads & links

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Map

FileDownload / File: emd_47049.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTom1 ubiquitylation cascade, closed-ring, consensus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 240 pix.
= 316.8 Å		1.32 Å/pix.
x 240 pix.
= 316.8 Å		1.32 Å/pix.
x 240 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.1563821 - 0.542901
Average (Standard dev.)-0.00019777074 (±0.023403019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 316.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Tom1 ubiquitylation cascade, closed-ring, half map B

Fileemd_47049_half_map_1.map
AnnotationTom1 ubiquitylation cascade, closed-ring, half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tom1 ubiquitylation cascade, closed-ring, half map A

Fileemd_47049_half_map_2.map
AnnotationTom1 ubiquitylation cascade, closed-ring, half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tom1 ubiquitylation cascade, closed-ring conformation

EntireName: Tom1 ubiquitylation cascade, closed-ring conformation
Components
  • Complex: Tom1 ubiquitylation cascade, closed-ring conformation
    • Protein or peptide: E3 ubiquitin-protein ligase Tom1

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Supramolecule #1: Tom1 ubiquitylation cascade, closed-ring conformation

SupramoleculeName: Tom1 ubiquitylation cascade, closed-ring conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complex identified by plunge-freezing Tom1 in active ubiquitylation cascade
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 440 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 ILDRMDGLL LKVAEKYKYK QDPKKECEVK LVEMEAHDVD YCLKMLKFTR RLLLNTENRF VYSSGDVLMY LLNCPNFTIK L AVMRILAI ...String:
MDWSHPQFEK SAVDENLYFQ GGGRMVLFTR CEKARKEKLA AGYKPLVDYL IDCDTPTFLE RIEAIQEWDR SRDDLYVWIP ILDRMDGLL LKVAEKYKYK QDPKKECEVK LVEMEAHDVD YCLKMLKFTR RLLLNTENRF VYSSGDVLMY LLNCPNFTIK L AVMRILAI LGERFVIARE KIVAHNIFGD HNLRKKTLKL ALSLSSSVMD EDGEHFSLVD LYFDKKKVPQ KWRKLRFTHY TS NDFKKSS QQKNNINETQ TSIKKVTMTT QELCEHSLQQ IFDKGMALLP AESWFDFSIK ASVAKAFSDD SGENIDLRNI IIE TKLNAI AFVNTIFSPP QVSSKLFELD PYAFNSLTDL ISLSETKIPK ELRTDALFTL ECISLKHVWC SDIIRNLGGN ISHG LLFQI LRYIAKTLRE ATDEIDEEYN VRFFYLISNL ADVKPLHESL FAAGLIPTLL EIVSIRNCPY KRTLASATHL LETFI DNSE TTTEFIENDG FTMLITSVAN EIDFTLAHPE TWQPPKYSVV YYSISFRELA YIRSLLKLVL KLLSTDSGDR IRNLID SPI LVSLKKILEN KLVFGLTLIT YTLDVVQKVI NSEPTIYPVL VEAGLIPYVI DNFPKLIGPS AELLSLLPDV VSAICLN PE GLKQVKEKGL INNLFDFLLD ADHARILTGG DRSTEYGTDI DELARHYPDL KANIVEALCN VIRKMPSTFR NEREFLFT S PKDQKYFFHR KNEEILTDKE EHEPAYWELL DKGTMLDTFT SVLFGMSLGN GSFSQVPQHL EARDFLAIIF MENPPYEYF TSVAISNVTE VLQYLDEKYE DYAFMDVMKV LNDQLENLND FLNSPNDRSF FLERDGENSV RSCHSKLCRL AAILNIVTNV YIDLTTLSC KRIMQIYSYF DKRGFSLIKN LKLLFQKCAL EEMYIRQHMP DSVITETMPL PIVDVSGDGP PLQIYIDDPK K GDQKGKIT SVKTRNTLQM RTILYTLQSN TAILFRCFLR LSHSRNMDLE HKDLTTEVHI FENVVENVIE MLKATELEGH LP YILVLLN FNTFVFTIPK ASPNSTEILQ TIPAYIFYQK GGYLLYLHII RDLFTRMTKI KDLSSLDNIN YIDESNGILT LSC LINALT FYNKSMQTET MENVQSIGKY YVSIDDDYNI MKALTVPIKV MALAMILDLD KSDSLFKTQS RNVPYSVFKQ LLSM LKNIF TNVNIYTKEL YELHWDLIFP PIKKISLFEQ VGIPGDVAAN YLTDTGDDLP ADNSIGLFSP EQWEKYKKLI GEDKS IYYP QPMQAQYYKG CSSKELDELR DTFFNDGLPS RIFTVLPFYP KLVNAFAKTL LQIFTKYDEP TEVFAGRILD RILETD LDD PATLSSLIHL FGIFLNEKYI YQKASHLMQR FIEYLEKSLK PEHVNTPWFS KALYVYEIIL AKSELPHLEE LSKDVLL RY PLLSMAKVFR IPDPMKQKLF DILIRVSDIS NFYSALATSR ILIFYSRDEL YANNIARSGI LSRLLKVIGS FQKLDKIN F LESSFLLLTR RCFETTENVD ALIRAEINRS FTARPLGGGD DAVRELTTIL EEKAHVVMRS PSQFIDVLCE TARFHEFDD QGALVDYSLK RFLGEKDKNT QASSTEKSDI YERTGIMHLL LSQLMAASEK DWLSEPANSS DLPENKKAQL DPSRNPVCAY MIFLLKLLV ELVSSYNQCK FEFLTFSRRN TYAERPRPRT TAINFFLYRL LDKPVGTDHD KHEAKRREVI GMLARSVIIG F LATVQDDR TTKTDVKLAD PHMNFIRKFA IEAIIKAIRN ATSSSKLLES NHLKLDMWFR IITSMVYVQA PYLRQLLDSN KV EADQYQL CKLVIDLGLP SVITEAMASI DLNYPFSKKI FNVAVEALNT ISSTRNNFSE HFKIEDHDEV EDEVDESDKE EIP DMFKNS ALGMYDVEDI EEDDDDDTSL IGDDDAMAFV DSDNGFEVVF SDEDDDMGEE DADDARSDSE ENELSSEMQS STAD GTDVD YEVDDADGLI INIDQPSGDD EEMADYDANI SHSSHSENED DASMDVIEVY DDELSSGYDV DLSDYDVDES DWDSG LSSL SISDEDSESS EDEPINSTRM GDSRRRWLIA EGVELTDDSQ GESEEDDRGV FRGIEHIFSN ENEPLFRVHD EMRHRN HHR SINRTHFHSA MSAPSLSLLN RGRRNQSNLI NPLGPTGLEQ VENDISDQVT VAGSGSRPRS HHLHFSEVLV SGSFFDE PV LDGIILKSTV SRWKDIFDMF YDSKTYANCI IPTVINRLYK VSLALQKDLE NKREQEKLKN KNLLFNEAKV ESHNSSDA I SVEQDDIQES NVTHDDHEPV YVTIQGSEVD IGGTDIDPEF MNALPDDIRA DVFAQHVRER RAEARLNSDH NVHSREIDS DFLEAIPEDI REGILDTEAE EQRMFGRIGS SADVIRADDD VSNNDEEVEN GLDHGNSNDR NNADPEKKKP ARIYFAPLID RAGIASLMK SVFISKPYIQ REIYHELFYR LCSSKQNRND LMNTFLFILS EGIIDQHSLE KVYNIISSRA MGHAKTTTVR Q LPSDCTPL TVANQTIEIL QSLIDADSRL KYFLIAEHDN LIVNKANNKS RKEALPDKKL RWPLWHLFSL LDRKLITDES VL MDLLTRI LQVCTKTLAV LSTSSNGKEN LSKKFHLPSF DEDDLMKILS IIMLDSCTTR VFQQTLNIIY NLSKLQGCMS IFT KHLVSL AISIMSKLKS ALDGLSREVG TITTGMEINS ELLQKFTLPS SDQAKLLKIL TTVDFLYTHK RKEEERNVKD LQSL YDKMN GGPVWSSLSE CLSQFEKSQA INTSATILLP LIESLMVVCR RSDLSQNRNT AVKYEDAKLL DFSKTRVENL FFPFT DAHK KLLNQMIRSN PKLMSGPFAL LVKNPKVLDF DNKRYFFNAK LKSDNQERPK LPITVRREQV FLDSYRALFF KTNDEI KNS KLEITFKGES GVDAGGVTRE WYQVLSRQMF NPDYALFLPV PSDKTTFHPN RTSGINPEHL SFFKFIGMII GKAIRDQ CF LDCHFSREVY KNILGRPVSL KDMESLDPDY YKSLVWILEN DITDIIEETF SVETDDYGEH KVINLIEGGK DIIVTEAN K QDYVKKVVEY KLQTSVKEQM DNFLVGFYAL ISKDLITIFD EQELELLISG LPDIDVDDWK NNTTYVNYTA TCKEVSYFW RAVRSFDAEE RAKLLQFVTG TSKVPLNGFK ELSGVNGVCK FSIHRDFGSS ERLPSSHTCF NQLNLPPYES YETLRGSLLL AINEGHEGF GLA

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

Concentration1.9 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
15.0 mMHEPESHEPES
1.0 mMTCEPtris(2-carboxyethyl)phosphine
0.1 mMCHAPSOCHAPSO
1.0 mMMg-ATPMagnesium Adenosine triphosphate
0.5 XCommercialE3 Ligase Reaction Buffer (R&D Systems)

Details: 1.5 mM HEPES pH 7.2, 100 mM NaCl, 1 mM TCEP, 0.1 mM CHAPSO, 1 mM Mg-ATP, 0.5X E3 Ligase Reaction Buffer (R and D biosystems)
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: 283.15 K / Instrument: LEICA PLUNGER
Details: CHAPSO detergent added to final conc. of 0.1 mM. Sample applied twice..
DetailsUBA1 and UBE2D2 were pre-charged for 20 minutes at room temperature in a solution containing 0.5 uM UBA1, 18 uM UBE2D2, 2 mM Mg-ATP (R and D Systems) and 25 uM ubiquitin (R and D Systems), buffered with 1X E3 Ligase Reaction Buffer (R and D Systems). Separately, Tom1 was pre-incubated with histone H2B at room temperature at room temperature for 20 minutes at a concentration of 8 uM Tom1 and 16 uM histone H2B. Samples were pre-mixed at a one-to-one volume-volume ratio for 45 seconds and 90 seconds, before addition of detergent and immediate sample application (final sample concentration: 4 uM Tom1, 8 uM histone H2B, 0.25 uM UBA1, 9 uM UBE2D2, 1 mM Mg-ATP, 12.5 uM ubiquitin, 0.2 mM CHAPSO).

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8592 / Average exposure time: 4.993 sec. / Average electron dose: 52.55 e/Å2
Electron beamAcceleration voltage: 200 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: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

Particle selectionNumber selected: 3159343
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Data collected from previous EM session
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 496436
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 modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model

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