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- EMDB-27274: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubi... -

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Basic information

Entry
Database: EMDB / ID: EMD-27274
TitleSaccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
Map datacomposite map of the substrate interacting state 'intA'
Sample
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
      • 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 ubiquitin moieties bound to the Ufd1/Npl4 tower
      • 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 / endoplasmic reticulum membrane fusion / Cdc48p-Npl4p-Vms1p AAA ATPase complex / Doa10p ubiquitin ligase complex / DNA replication termination / stress-induced homeostatically regulated protein degradation pathway / positive regulation of mitochondrial fusion / sister chromatid biorientation / ribophagy ...SCF complex disassembly in response to cadmium stress / Ovarian tumor domain proteases / endoplasmic reticulum membrane fusion / Cdc48p-Npl4p-Vms1p AAA ATPase complex / Doa10p ubiquitin ligase complex / DNA replication termination / stress-induced homeostatically regulated protein degradation pathway / positive regulation of mitochondrial fusion / sister chromatid biorientation / ribophagy / 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 / Translesion Synthesis by POLH / piecemeal microautophagy of the nucleus / mating projection tip / Protein methylation / vesicle-fusing ATPase / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / replisome / ribosome-associated ubiquitin-dependent protein catabolic process / nuclear outer membrane-endoplasmic reticulum membrane network / retrograde protein transport, ER to cytosol / KEAP1-NFE2L2 pathway / Neddylation / protein quality control for misfolded or incompletely synthesized proteins / autophagosome maturation / ribosomal large subunit export from nucleus / polyubiquitin modification-dependent protein binding / mRNA transport / ERAD pathway / ATP metabolic process / Neutrophil degranulation / rescue of stalled ribosome / ubiquitin binding / macroautophagy / ribosomal large subunit assembly / modification-dependent protein catabolic process / positive regulation of protein localization to nucleus / protein tag activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / nuclear membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / cytosolic large ribosomal subunit / cytoplasmic translation / protein ubiquitination / structural constituent of ribosome / ubiquitin protein ligase binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol
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 / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e 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 domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / 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
Ubiquitin-ribosomal protein eL40A fusion protein / 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: 3.5 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_27274.png

Downloads & links

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Map

FileDownload / File: emd_27274.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of the substrate interacting state 'intA'
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 9.0
Minimum - Maximum-27.384119 - 68.013306
Average (Standard dev.)-0.016353576 (±1.0786074)
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: post-processed overall refinement map of the substrate interacting...

Fileemd_27274_additional_1.map
Annotationpost-processed overall refinement map of the substrate interacting state 'intA'
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 upper Npl4 with...

Fileemd_27274_additional_10.map
Annotationpost-processed focused refinement map of upper Npl4 with polyubiquitin of the substrate interacting state 'intA'
Projections & Slices
AxesZYX

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

Fileemd_27274_additional_11.map
Annotationpost-processed focused refinement map of the Ufd1/Npl4 tower with polyubiquitin of the substrate interacting state 'intA'
Projections & Slices
AxesZYX

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Additional map: post-processed focused refinement map of cdc48 of the...

Fileemd_27274_additional_12.map
Annotationpost-processed focused refinement map of cdc48 of the substrate interacting state 'intA'
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Additional map: focused refinement half map 1 of cdc48 of...

Fileemd_27274_additional_13.map
Annotationfocused refinement half map 1 of cdc48 of the substrate interacting state 'intA'
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Additional map: focused refinement half map 2 of cdc48 of...

Fileemd_27274_additional_2.map
Annotationfocused refinement half map 2 of cdc48 of the substrate interacting state 'intA'
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Additional map: focused refinement half map 1 of the Ufd1/Npl4...

Fileemd_27274_additional_3.map
Annotationfocused refinement half map 1 of the Ufd1/Npl4 tower with polyubiquitin of the substrate interacting state 'intA'
Projections & Slices
AxesZYX

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Additional map: focused refinement half map 2 of upper Npl4...

Fileemd_27274_additional_4.map
Annotationfocused refinement half map 2 of upper Npl4 with polyubiquitin of the substrate interacting state 'intA'
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AxesZYX

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Additional map: focused refinement half map 2 of the Ufd1/Npl4...

Fileemd_27274_additional_5.map
Annotationfocused refinement half map 2 of the Ufd1/Npl4 tower with polyubiquitin of the substrate interacting state 'intA'
Projections & Slices
AxesZYX

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Histogram

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

Fileemd_27274_additional_6.map
Annotationfocused refinement half map 1 of upper Npl4 with polyubiquitin of the substrate interacting state 'intA'
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AxesZYX

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

Fileemd_27274_additional_7.map
Annotationpost-processed focused refinement map of D1/D2 domains of cdc48 of the substrate interacting state 'intA'
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AxesZYX

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

Fileemd_27274_additional_8.map
Annotationfocused refinement half map 1 of D1/D2 domains of cdc48 of the substrate interacting state 'intA'
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Additional map: focused refinement half map 2 of D1/D2 domains...

Fileemd_27274_additional_9.map
Annotationfocused refinement half map 2 of D1/D2 domains of cdc48 of the substrate interacting state 'intA'
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AxesZYX

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Half map: overall refinement half map of the substrate interacting state 'intA'

Fileemd_27274_half_map_1.map
Annotationoverall refinement half map of the substrate interacting state 'intA'
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Half map: overall refinement half map 2 of the substrate...

Fileemd_27274_half_map_2.map
Annotationoverall refinement half map 2 of the substrate interacting state 'intA'
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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 in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
Components
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
      • 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 ubiquitin moieties bound to the Ufd1/Npl4 tower
      • 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 in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Details: Ufd1/Npl4/Cdc48 was pre-incubated with polyubiquitylated SUMO-ubiquitin-mEOS and ATP - this class represents the portion of the particles where the complex is interacting with two folded ...Details: Ufd1/Npl4/Cdc48 was pre-incubated with polyubiquitylated SUMO-ubiquitin-mEOS and ATP - this class represents the portion of the particles where the complex is interacting with two folded ubiquitin moieties (state intA)
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 in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#4
Details: overall refinement map is "uncclosedE_overall_291_postprocess_masked.mrc" from half maps "uncclosedE_overall_half1.mrc" and "uncclosedE_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 "uncclosedE_cdc48_502_postprocess_masked.mrc" from half maps "uncclosedE_cdc48_half1.mrc" and "uncclosedE_cdc48_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #4: Two folded ubiquitin moieties bound to the Ufd1/Npl4 tower

SupramoleculeName: Two folded ubiquitin moieties bound to the Ufd1/Npl4 tower
type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #2-#4
Details: focused refinement map of the Ufd1/Npl4 tower with polyubiquitin is "uncclosedE_tower_551_postprocess_masked.mrc" from half maps "uncclosedE_tower_half1.mrc" and "uncclosedE_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 domains of Cdc48 is "uncclosedE_atpase_535_postprocess_masked.mrc" from half maps "uncclosedE_atpase_half1.mrc" and "uncclosedE_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 upper Npl4 with polyubiquitin is "uncclosedE_ub_556_postprocess_masked.mrc" from half maps "uncclosedE_ub_half1.mrc" and "uncclosedE_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 ubiquitin with a K48-linked polyubiquitin. Sequence of the unmodified substrate ...Details: ubiquitin that is part of the SUMO-ubiquitin(K48polyUb)-mEOS substrate; SUMO-ubiquitin-mEOS modified on K48 of ubiquitin with a K48-linked polyubiquitin. Sequence of the unmodified substrate is MGSSHHHHHHSSGENLYFQGHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSHMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVKEGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNARNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLEGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPDNARR
Number of copies: 2 / 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

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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

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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.
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 70.577 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.2 µm
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
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 29595
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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