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- EMDB-47046: Focus map, Cryo-EM map of Tom1-UBE2D2-ubiquitin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-47046
TitleFocus map, Cryo-EM map of Tom1-UBE2D2-ubiquitin complex
Map dataconsensus map, Tom1-E2-ubiquitin focus map
Sample
  • Complex: Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ubiquitin conjugating enzyme UBE2D2 and ubiquitin
    • Protein or peptide: E3 ubiquitin-protein ligase Tom1
    • Protein or peptide: E2 ubiquitin-conjugating enzyme UBE2D2
    • Protein or peptide: Polyubiquitin-C
Keywordstransferase / ubiquitin / ubiquitylation complex / ligase
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) / (E3-independent) E2 ubiquitin-conjugating enzyme / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / E2 ubiquitin-conjugating enzyme / 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 / ubiquitin conjugating enzyme activity ...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) / (E3-independent) E2 ubiquitin-conjugating enzyme / HECT-type E3 ubiquitin transferase / nucleocytoplasmic transport / E2 ubiquitin-conjugating enzyme / 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 / ubiquitin conjugating enzyme activity / Antigen processing: Ubiquitination & Proteasome degradation / mRNA transport / protein K48-linked ubiquitination / 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) / protein autoubiquitination / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Neutrophil degranulation / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Constitutive Signaling by NOTCH1 HD Domain Mutants / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / APC-Cdc20 mediated degradation of Nek2A / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / Regulation of innate immune responses to cytosolic DNA / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / NF-kB is activated and signals survival / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / NRIF signals cell death from the nucleus / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / Translesion synthesis by POLK / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / InlB-mediated entry of Listeria monocytogenes into host cell / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Translesion synthesis by POLI / Regulation of activated PAK-2p34 by proteasome mediated degradation / IKK complex recruitment mediated by RIP1 / Gap-filling DNA repair synthesis and ligation in GG-NER / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / TCF dependent signaling in response to WNT / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Regulation of NF-kappa B signaling / Asymmetric localization of PCP proteins / Ubiquitin-dependent degradation of Cyclin D / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / activated TAK1 mediates p38 MAPK activation / Negative regulators of DDX58/IFIH1 signaling / TNFR2 non-canonical NF-kB pathway / AUF1 (hnRNP D0) binds and destabilizes mRNA / Regulation of signaling by CBL / NOTCH3 Activation and Transmission of Signal to the Nucleus / Vpu mediated degradation of CD4 / Assembly of the pre-replicative complex / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Degradation of DVL / Deactivation of the beta-catenin transactivating complex / Negative regulation of FGFR3 signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Dectin-1 mediated noncanonical NF-kB signaling / Fanconi Anemia Pathway / Peroxisomal protein import / Degradation of AXIN / Regulation of TNFR1 signaling
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 / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Armadillo-like helical / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Armadillo-type fold / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Polyubiquitin-C / Ubiquitin-conjugating enzyme E2 D2 / E3 ubiquitin-protein ligase TOM1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsWarner KM / Hunkeler M / Baek K / Roy Burman SS / 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_47046.png

Downloads & links

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Map

FileDownload / File: emd_47046.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationconsensus map, Tom1-E2-ubiquitin 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: 0.02
Minimum - Maximum-0.06294887 - 0.17543219
Average (Standard dev.)0.00025132223 (±0.0044443337)
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_47046_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Mask #2

Fileemd_47046_msk_2.map
Projections & Slices
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Additional map: DeepEMhancer sharpened map, Tom1-E2-ubiquitin focus map

Fileemd_47046_additional_1.map
AnnotationDeepEMhancer sharpened map, Tom1-E2-ubiquitin focus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map A, Tom1-E2-ubiquitin focus map

Fileemd_47046_half_map_1.map
Annotationhalf map A, Tom1-E2-ubiquitin 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-E2-ubiquitin focus map

Fileemd_47046_half_map_2.map
Annotationhalf map B, Tom1-E2-ubiquitin focus map
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ub...

EntireName: Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ubiquitin conjugating enzyme UBE2D2 and ubiquitin
Components
  • Complex: Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ubiquitin conjugating enzyme UBE2D2 and ubiquitin
    • Protein or peptide: E3 ubiquitin-protein ligase Tom1
    • Protein or peptide: E2 ubiquitin-conjugating enzyme UBE2D2
    • Protein or peptide: Polyubiquitin-C

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Supramolecule #1: Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ub...

SupramoleculeName: Focus Map, Ternary complex of E3 ubiquitin ligase Tom1 with E2 ubiquitin conjugating enzyme UBE2D2 and ubiquitin
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: 444 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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Macromolecule #2: E2 ubiquitin-conjugating enzyme UBE2D2

MacromoleculeName: E2 ubiquitin-conjugating enzyme UBE2D2 / type: protein_or_peptide / ID: 2 / Details: TEV cleavage product GST-TEV-UBE2D2 / Enantiomer: LEVO / EC number: ubiquitin-protein ligase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMALKRIHK ELNDLARDPP AQCSAGPVGD DMFHWQATIM GPNDSPYQGG VFFLTIHFPT DYPFKPPKVA FTTRIYHPNI NSNGSICLD ILRSQWSPAL TISKVLLSIC SLLCDPNPDD PLVPEIARIY KTDREKYNRI AREWTQKYAM

UniProtKB: Ubiquitin-conjugating enzyme E2 D2

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Macromolecule #3: Polyubiquitin-C

MacromoleculeName: Polyubiquitin-C / type: protein_or_peptide / ID: 3 / Details: Ubiquitin / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

UniProtKB: Polyubiquitin-C

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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
200.0 mMNaClsodium chloride
30.0 mMHEPESHEPES
0.5 mMTCEPtris(2-carboxyethyl)phosphine
0.2 mMCHAPSOCHAPSO

Details: 30 mM HEPES pH 7.2, 200 mM NaCl, 0.5 mM TCEP, 0.2 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 0.2 mM. Sample applied twice..
DetailsSample was prepared by pre-forming Tom1:H2B complex. In tandem, UBE2D2/UBA1/ubiquitin were incubated in reaction buffer containing Mg-ATP for twenty minutes at room temperature, then buffer exchanged. The enzymatic cascade was initiated by mixing both samples and quenched by plunge freezing. Final sample concentrations were: 5 uM Tom1, 10 uM histone H2B, 0.25 uM UBA1, 44 uM UBE2D2, 75 uM ubiquitin, 0.2 mM CHAPSO.

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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: 18959 / Average exposure time: 2.896 sec. / Average electron dose: 56.13 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: 0.8 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: 11388836
Details: Initial 2D classes assisted template picking in cryoSPARC Live.
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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 246341
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC (ver. 4.4.1) / Details: heterogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3280

source_name: AlphaFold, initial_model_type: in silico model

4v3l

chain_id: A, source_name: PDB, initial_model_type: experimental model

4v3l

chain_id: B, source_name: PDB, initial_model_type: experimental model
DetailsPhenix real-space refinement
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 89.6 / Target criteria: Cross-correlation coefficient

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  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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