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- EMDB-27276: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two fol... -

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Basic information

Entry
Database: EMDB / ID: EMD-27276
TitleSaccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
Map datacomposite map of the ubiquitin unfolded state 'uA'
Sample
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubiquitin moieties and one unfolded Ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
      • Protein or peptide: Cell division control protein 48
      • Protein or peptide: Nuclear protein localization protein 4
      • Protein or peptide: Ubiquitin fusion degradation protein 1
      • Protein or peptide: Ubiquitin
    • Complex: Cdc48 hexamer
      • Complex: D1/D2 ATPase domains of the Cdc48 hexamer
    • Complex: Two folded ubiqutin moieties and one unfolded ubiquitin bound to Ufd1/Npl4
      • Complex: Two folded ubiqutin moieties bound to Npl4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
Function / homology
Function and homology information


SCF complex disassembly in response to cadmium stress / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / endoplasmic reticulum membrane fusion / Cdc48p-Npl4p-Vms1p AAA ATPase complex / Doa10p ubiquitin ligase complex / ribophagy / DNA replication termination / Neddylation / stress-induced homeostatically regulated protein degradation pathway ...SCF complex disassembly in response to cadmium stress / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / endoplasmic reticulum membrane fusion / Cdc48p-Npl4p-Vms1p AAA ATPase complex / Doa10p ubiquitin ligase complex / ribophagy / DNA replication termination / Neddylation / stress-induced homeostatically regulated protein degradation pathway / positive regulation of mitochondrial fusion / sister chromatid biorientation / cytoplasm protein quality control by the ubiquitin-proteasome system / Hrd1p ubiquitin ligase ERAD-L complex / RQC complex / protein localization to vacuole / protein-containing complex disassembly / mitochondria-associated ubiquitin-dependent protein catabolic process / nuclear protein quality control by the ubiquitin-proteasome system / HSF1 activation / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / nonfunctional rRNA decay / protein phosphatase regulator activity / piecemeal microautophagy of the nucleus / mating projection tip / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / replisome / vesicle-fusing ATPase / : / ribosome-associated ubiquitin-dependent protein catabolic process / retrograde protein transport, ER to cytosol / nuclear outer membrane-endoplasmic reticulum membrane network / protein quality control for misfolded or incompletely synthesized proteins / autophagosome maturation / polyubiquitin modification-dependent protein binding / mRNA transport / rescue of stalled ribosome / : / ATP metabolic process / Neutrophil degranulation / ubiquitin binding / macroautophagy / modification-dependent protein catabolic process / protein tag activity / positive regulation of protein localization to nucleus / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / nuclear membrane / protein ubiquitination / ubiquitin protein ligase binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / Ubiquitin fusion degradation protein UFD1 / NPL4, zinc-binding putative / Nuclear protein localization protein 4 / NPL4 family, putative zinc binding region / Nuclear pore localisation protein NPL4, C-terminal / NPL4 family / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain ...Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / Ubiquitin fusion degradation protein UFD1 / NPL4, zinc-binding putative / Nuclear protein localization protein 4 / NPL4 family, putative zinc binding region / Nuclear pore localisation protein NPL4, C-terminal / NPL4 family / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / MPN domain / MPN domain profile. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Polyubiquitin / Cell division control protein 48 / Nuclear protein localization protein 4 / Ubiquitin fusion degradation protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLee HG / Lima CD
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118080 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: SUMO enhances unfolding of SUMO-polyubiquitin-modified substrates by the Ufd1/Npl4/Cdc48 complex.
Authors: Hyein G Lee / Abigail A Lemmon / Christopher D Lima /
Abstract: The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets ...The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets are established; however, prior studies suggest that the complex also targets substrates modified by the ubiquitin-like protein SUMO. Here, we show that interactions between Ufd1 and SUMO enhance unfolding of substrates modified by SUMO-polyubiquitin hybrid chains by the budding yeast Ufd1/Npl4/Cdc48 complex compared to substrates modified by polyubiquitin chains, a difference that is accentuated when the complex has a choice between these substrates. Incubating Ufd1/Npl4/Cdc48 with a substrate modified by a SUMO-polyubiquitin hybrid chain produced a series of single-particle cryo-EM structures that reveal features of interactions between Ufd1/Npl4/Cdc48 and ubiquitin prior to and during unfolding of ubiquitin. These results are consistent with cellular functions for SUMO and ubiquitin modifications and support a physical model wherein Ufd1/Npl4/Cdc48, SUMO, and ubiquitin conjugation pathways converge to promote clearance of proteins modified with SUMO and polyubiquitin.
History
DepositionJun 14, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27276.png

Downloads & links

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Map

FileDownload / File: emd_27276.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of the ubiquitin unfolded state 'uA'
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 9.5
Minimum - Maximum-23.553677 - 66.1101
Average (Standard dev.)-0.016668871 (±1.1007812)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 408.576 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: overall refinement post-processed map of the ubiquitin unfolded...

Fileemd_27276_additional_1.map
Annotationoverall refinement post-processed map of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Additional map: post-processed focused refinement map of the D1/D2 domains...

Fileemd_27276_additional_10.map
Annotationpost-processed focused refinement map of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Additional map: focused refinement half map 1 of Cdc48 of...

Fileemd_27276_additional_11.map
Annotationfocused refinement half map 1 of Cdc48 of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Additional map: post-processed focused refinement map of Npl4 and polyubiquitin...

Fileemd_27276_additional_12.map
Annotationpost-processed focused refinement map of Npl4 and polyubiquitin density of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Additional map: focused refinement half map 1 of Npl4 and...

Fileemd_27276_additional_13.map
Annotationfocused refinement half map 1 of Npl4 and polyubiquitin density of the ubiquitin unfolded state 'uA'
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Additional map: focused refinement half map 2 of the D1/D2...

Fileemd_27276_additional_2.map
Annotationfocused refinement half map 2 of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Additional map: focused refinement half map 1 of the D1/D2...

Fileemd_27276_additional_3.map
Annotationfocused refinement half map 1 of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uA'
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Additional map: focused refinement half map 1 of the central...

Fileemd_27276_additional_4.map
Annotationfocused refinement half map 1 of the central Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uA'
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AxesZYX

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Additional map: focused refinement half map 2 of Npl4/polyubiquitin density...

Fileemd_27276_additional_5.map
Annotationfocused refinement half map 2 of Npl4/polyubiquitin density of the ubiquitin unfolded state 'uA'
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Additional map: focused refinement half map 2 of Cdc48 of...

Fileemd_27276_additional_6.map
Annotationfocused refinement half map 2 of Cdc48 of the ubiquitin unfolded state 'uA'
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Additional map: focused refinement half map 2 of the central...

Fileemd_27276_additional_7.map
Annotationfocused refinement half map 2 of the central Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Histogram

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Additional map: post-processed focused refinement map of Cdc48 of the...

Fileemd_27276_additional_8.map
Annotationpost-processed focused refinement map of Cdc48 of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: post-processed focused refinement map of the central Ufd1/Npl4/polyubiquitin...

Fileemd_27276_additional_9.map
Annotationpost-processed focused refinement map of the central Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uA'
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: overall refinement half map 1

Fileemd_27276_half_map_1.map
Annotationoverall refinement half map 1
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Half map: overall refinement half map 2 of the ubiquitin unfolded state 'uA'

Fileemd_27276_half_map_2.map
Annotationoverall refinement half map 2 of the ubiquitin unfolded state 'uA'
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Sample components

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Entire : Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubi...

EntireName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubiquitin moieties and one unfolded Ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
Components
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubiquitin moieties and one unfolded Ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
      • Protein or peptide: Cell division control protein 48
      • Protein or peptide: Nuclear protein localization protein 4
      • Protein or peptide: Ubiquitin fusion degradation protein 1
      • Protein or peptide: Ubiquitin
    • Complex: Cdc48 hexamer
      • Complex: D1/D2 ATPase domains of the Cdc48 hexamer
    • Complex: Two folded ubiqutin moieties and one unfolded ubiquitin bound to Ufd1/Npl4
      • Complex: Two folded ubiqutin moieties bound to Npl4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubi...

SupramoleculeName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two Ubiquitin moieties and one unfolded Ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Details: Composite map. Ufd1/Npl4/Cdc48 was pre-incubated with polyubiquitylated SUMO-ubiquitin-mEOS and ATP - this class represents the portion of the substrate-bound complex engaged with two folded ...Details: Composite map. Ufd1/Npl4/Cdc48 was pre-incubated with polyubiquitylated SUMO-ubiquitin-mEOS and ATP - this class represents the portion of the substrate-bound complex engaged with two folded ubiquitin moieties and one unfolded ubiquitin (state uA)
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #2: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubi...

SupramoleculeName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (uA)
type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#4
Details: overall refinement map is "uncopenB_overall_388_postprocess_masked.mrc" and associated half maps are "uncopen_overall_half1.mrc" and "uncopen_overall_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: Cdc48 hexamer

SupramoleculeName: Cdc48 hexamer / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Details: focused refinement map of the Cdc48 hexamer is "uncopenB_cdc48_525_postprocess_masked.mrc" and associated half maps are "uncopenB_cdc48_half1.mrc" and "uncopenB_cdc48_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #4: Two folded ubiqutin moieties and one unfolded ubiquitin bound to ...

SupramoleculeName: Two folded ubiqutin moieties and one unfolded ubiquitin bound to Ufd1/Npl4
type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #2-#4
Details: focused refinement map of Npl4/polyubiquitin density is "uncopenB_tower_484_postprocess_masked.mrc" and associated half maps are "uncopenB_tower_half1.mrc" and "uncopenB_tower_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #5: D1/D2 ATPase domains of the Cdc48 hexamer

SupramoleculeName: D1/D2 ATPase domains of the Cdc48 hexamer / type: complex / ID: 5 / Chimera: Yes / Parent: 3 / Macromolecule list: #1
Details: focused refinement map of the D1/D2 ATPase domains of Cdc48 is "uncopenB_atpase_531_postprocess_masked.mrc" and associated half maps are "uncopenB_atpase_half1.mrc" and "uncopenB_atpase_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #6: Two folded ubiqutin moieties bound to Npl4

SupramoleculeName: Two folded ubiqutin moieties bound to Npl4 / type: complex / ID: 6 / Chimera: Yes / Parent: 4 / Macromolecule list: #2, #4
Details: focused refinement map of the upper density of Npl4/polyubiquitin is "uncopenB_ub_489_postprocess_masked.mrc" and associated half maps are "uncopenB_ub_half1.mrc" and "uncopenB_ub_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Cell division control protein 48

MacromoleculeName: Cell division control protein 48 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 92.389195 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMGEEHKP LLDASGVDPR EEDKTATAIL RRKKKDNMLL VDDAINDDNS VIAINSNTMD KLELFRGDTV LVKGKKRKDT VLIVLIDDE LEDGACRINR VVRNNLRIRL GDLVTIHPCP DIKYATRISV LPIADTIEGI TGNLFDVFLK PYFVEAYRPV R KGDHFVVR ...String:
GSHMGEEHKP LLDASGVDPR EEDKTATAIL RRKKKDNMLL VDDAINDDNS VIAINSNTMD KLELFRGDTV LVKGKKRKDT VLIVLIDDE LEDGACRINR VVRNNLRIRL GDLVTIHPCP DIKYATRISV LPIADTIEGI TGNLFDVFLK PYFVEAYRPV R KGDHFVVR GGMRQVEFKV VDVEPEEYAV VAQDTIIHWE GEPINREDEE NNMNEVGYDD IGGCRKQMAQ IREMVELPLR HP QLFKAIG IKPPRGVLMY GPPGTGKTLM ARAVANETGA FFFLINGPEV MSKMAGESES NLRKAFEEAE KNAPAIIFID EID SIAPKR DKTNGEVERR VVSQLLTLMD GMKARSNVVV IAATNRPNSI DPALRRFGRF DREVDIGIPD ATGRLEVLRI HTKN MKLAD DVDLEALAAE THGYVGADIA SLCSEAAMQQ IREKMDLIDL DEDEIDAEVL DSLGVTMDNF RFALGNSNPS ALRET VVES VNVTWDDVGG LDEIKEELKE TVEYPVLHPD QYTKFGLSPS KGVLFYGPPG TGKTLLAKAV ATEVSANFIS VKGPEL LSM WYGESESNIR DIFDKARAAA PTVVFLDELD SIAKARGGSL GDAGGASDRV VNQLLTEMDG MNAKKNVFVI GATNRPD QI DPAILRPGRL DQLIYVPLPD ENARLSILNA QLRKTPLEPG LELTAIAKAT QGFSGADLLY IVQRAAKYAI KDSIEAHR Q HEAEKEVKVE GEDVEMTDEG AKAEQEPEVD PVPYITKEHF AEAMKTAKRS VSDAELRRYE AYSQQMKASR GQFSNFNFN DAPLGTTATD NANSNNSAPS GAGAAFGSNA EEDDDLYS

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Macromolecule #2: Nuclear protein localization protein 4

MacromoleculeName: Nuclear protein localization protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 66.144336 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMLIRFRS KNGTHRVSCQ ENDLFGTVIE KLVGNLDPNA DVDTFTVCEK PGQGIHAVSE LADRTVMDLG LKHGDMLILN YSDKPANEK DGVNVEIGSV GIDSKGIRQH RYGPLRIKEL AVDEELEKED GLIPRQKSKL CKHGDRGMCE YCSPLPPWDK E YHEKNKIK ...String:
GSHMLIRFRS KNGTHRVSCQ ENDLFGTVIE KLVGNLDPNA DVDTFTVCEK PGQGIHAVSE LADRTVMDLG LKHGDMLILN YSDKPANEK DGVNVEIGSV GIDSKGIRQH RYGPLRIKEL AVDEELEKED GLIPRQKSKL CKHGDRGMCE YCSPLPPWDK E YHEKNKIK HISFHSYLKK LNENANKKEN GSSYISPLSE PDFRINKRCH NGHEPWPRGI CSKCQPSAIT LQQQEFRMVD HV EFQKSEI INEFIQAWRY TGMQRFGYMY GSYSKYDNTP LGIKAVVEAI YEPPQHDEQD GLTMDVEQVK NEMLQIDRQA QEM GLSRIG LIFTDLSDAG AGDGSVFCKR HKDSFFLSSL EVIMAARHQT RHPNVSKYSE QGFFSSKFVT CVISGNLEGE IDIS SYQVS TEAEALVTAD MISGSTFPSM AYINDTTDER YVPEIFYMKS NEYGITVKEN AKPAFPVDYL LVTLTHGFPN TDTET NSKF VSSTGFPWSN RQAMGQSQDY QELKKYLFNV ASSGDFNLLH EKISNFHLLL YINSLQILSP DEWKLLIESA VKNEWE ESL LKLVSSAGWQ TLVMILQESG

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Macromolecule #3: Ubiquitin fusion degradation protein 1

MacromoleculeName: Ubiquitin fusion degradation protein 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 40.001449 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMFSGFSSF GGGNGFVNMP QTFEEFFRCY PIAMMNDRIR KDDANFGGKI FLPPSALSKL SMLNIRYPML FKLTANETGR VTHGGVLEF IAEEGRVYLP QWMMETLGIQ PGSLLQISST DVPLGQFVKL EPQSVDFLDI SDPKAVLENV LRNFSTLTVD D VIEISYNG ...String:
GSMFSGFSSF GGGNGFVNMP QTFEEFFRCY PIAMMNDRIR KDDANFGGKI FLPPSALSKL SMLNIRYPML FKLTANETGR VTHGGVLEF IAEEGRVYLP QWMMETLGIQ PGSLLQISST DVPLGQFVKL EPQSVDFLDI SDPKAVLENV LRNFSTLTVD D VIEISYNG KTFKIKILEV KPESSSKSIC VIETDLVTDF APPVGYVEPD YKALKAQQDK EKKNSFGKGQ VLDPSVLGQG SM STRIDYA GIANSSRNKL SKFVGQGQNI SGKAPKAEPK QDIKDMKITF DGEPAKLDLP EGQLFFGFPM VLPKEDEESA AGS KSSEQN FQGQGISLRK SNKRKTKSDH DSSKSKAPKS PEVIEID

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Macromolecule #4: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 4
Details: ubiquitin that is part of the SUMO-ubiquitin(K48polyUb)-mEOS substrate; SUMO-ubiquitin-mEOS modified on K48 of Ub with a K48-linked polyubiquitin. Sequence of the unmodified substrate is ...Details: ubiquitin that is part of the SUMO-ubiquitin(K48polyUb)-mEOS substrate; SUMO-ubiquitin-mEOS modified on K48 of Ub with a K48-linked polyubiquitin. Sequence of the unmodified substrate is MGSSHHHHHHSSGENLYFQGHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSHMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVKEGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNARNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLEGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPDNARR
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 8.568769 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVESS DTIDNVKSKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 5 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 7 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration2 mg/mL
BufferpH: 8
Details: 20 mM HEPES pH 8.0, 150 mM NaCl, 0.1 mM TCEP, 1 mM MgCl2, 5 mM ATP. Added 0.05% CHAPSO before vitrification.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: 8 s wait, 4 s blot before plunging.
DetailsUfd1/Npl4/Cdc48 was pre-incubated with SUMO-ubiquitin(K48polyUb)-mEOS and ATP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 70.577 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model from cryosparc
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 36688
FSC plot (resolution estimation)

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