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- EMDB-27277: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubi... -

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Basic information

Entry
Database: EMDB / ID: EMD-27277
TitleSaccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
Map datacomposite map of the ubiquitin unfolded state 'uC'
Sample
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
      • 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 Ufd1/Npl4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
KeywordsATPASE / ATPASE COMPLEX / UBIQUITIN / SUMO / SMT3 / QUALITY CONTROL / MOTOR PROTEIN
Function / homology
Function and homology information


Josephin domain DUBs / RAS processing / Regulation of PTEN localization / ER Quality Control Compartment (ERQC) / UCH proteinases / Interleukin-1 signaling / Aggrephagy / Pexophagy / PINK1-PRKN Mediated Mitophagy / Regulation of pyruvate metabolism ...Josephin domain DUBs / RAS processing / Regulation of PTEN localization / ER Quality Control Compartment (ERQC) / UCH proteinases / Interleukin-1 signaling / Aggrephagy / Pexophagy / PINK1-PRKN Mediated Mitophagy / Regulation of pyruvate metabolism / SCF complex disassembly in response to cadmium stress / mitotic DNA replication termination / Ovarian tumor domain proteases / Cdc48p-Npl4p-Vms1p AAA ATPase complex / Doa10p ubiquitin ligase complex / stress-induced homeostatically regulated protein degradation pathway / Peroxisomal protein import / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / protein localization to vacuole / sister chromatid biorientation / Hrd1p ubiquitin ligase ERAD-L complex / endoplasmic reticulum membrane fusion / ribophagy / DNA replication termination / RQC complex / Metalloprotease DUBs / Endosomal Sorting Complex Required For Transport (ESCRT) / mitochondria-associated ubiquitin-dependent protein catabolic process / positive regulation of mitochondrial fusion / cytoplasm protein quality control by the ubiquitin-proteasome system / HSF1 activation / nuclear protein quality control by the ubiquitin-proteasome system / protein-containing complex disassembly / E3 ubiquitin ligases ubiquitinate target proteins / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / endosome to plasma membrane protein transport / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / protein phosphatase regulator activity / Translesion Synthesis by POLH / piecemeal microautophagy of the nucleus / Termination of translesion DNA synthesis / Negative regulators of DDX58/IFIH1 signaling / mating projection tip / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Protein methylation / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / replisome / ribosome-associated ubiquitin-dependent protein catabolic process / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / nuclear outer membrane-endoplasmic reticulum membrane network / nonfunctional rRNA decay / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / Regulation of PTEN stability and activity / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / Neddylation / Formation of TC-NER Pre-Incision Complex / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Gap-filling DNA repair synthesis and ligation in TC-NER / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / protein quality control for misfolded or incompletely synthesized proteins / L13a-mediated translational silencing of Ceruloplasmin expression / Antigen processing: Ubiquitination & Proteasome degradation / Dual incision in TC-NER / polyubiquitin modification-dependent protein binding / autophagosome maturation / mRNA transport / Ub-specific processing proteases / ATP metabolic process / ERAD pathway / Neutrophil degranulation / rescue of stalled ribosome / ubiquitin binding / macroautophagy / modification-dependent protein catabolic process / positive regulation of protein localization to nucleus / protein tag activity / peroxisome / ubiquitin-dependent protein catabolic process / nuclear membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / protein ubiquitination / ubiquitin protein ligase binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding
Similarity search - Function
Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / : / : / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 1 / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 2 / NPL4, zinc-binding putative / NPL4 family, putative zinc binding region / Nuclear pore localisation protein NPL4, C-terminal / Nuclear protein localization protein 4 ...Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / : / : / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 1 / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 2 / NPL4, zinc-binding putative / NPL4 family, putative zinc binding region / Nuclear pore localisation protein NPL4, C-terminal / Nuclear protein localization protein 4 / 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 / MPN domain / MPN domain profile. / ATPase, AAA-type, conserved site / AAA-protein family signature. / 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
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: 3.5 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-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27277.png

Downloads & links

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Map

FileDownload / File: emd_27277.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of the ubiquitin unfolded state 'uC'
Projections & slices

Image control

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AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 384 pix.
= 408.576 Å		1.06 Å/pix.
x 384 pix.
= 408.576 Å		1.06 Å/pix.
x 384 pix.
= 408.576 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
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Contour LevelBy AUTHOR: 9.0
Minimum - Maximum-25.923601000000001 - 66.073740000000001
Average (Standard dev.)-0.01667898 (±1.0809361)
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-process overall refinement map of the ubiquitin unfolded...

Fileemd_27277_additional_1.map
Annotationpost-process overall refinement map of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 2 of the Cdc48...

Fileemd_27277_additional_10.map
Annotationfocused refinement half map 2 of the Cdc48 hexamer of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 1 of the D1/D2...

Fileemd_27277_additional_11.map
Annotationfocused refinement half map 1 of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 1 of the upper...

Fileemd_27277_additional_12.map
Annotationfocused refinement half map 1 of the upper Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 1 of the Cdc48...

Fileemd_27277_additional_13.map
Annotationfocused refinement half map 1 of the Cdc48 hexamer of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 2 of the upper...

Fileemd_27277_additional_2.map
Annotationfocused refinement half map 2 of the upper Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Additional map: post-processed focused refinement map of the Cdc48 hexamer...

Fileemd_27277_additional_3.map
Annotationpost-processed focused refinement map of the Cdc48 hexamer of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 2 of the D1/D2...

Fileemd_27277_additional_4.map
Annotationfocused refinement half map 2 of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uC'
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Additional map: post-processed focused refinement map of the D1/D2 domains...

Fileemd_27277_additional_5.map
Annotationpost-processed focused refinement map of the D1/D2 domains of Cdc48 of the ubiquitin unfolded state 'uC'
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Additional map: post-processed focused refinement map of the upper Ufd1/Npl4/polyubiquitin...

Fileemd_27277_additional_6.map
Annotationpost-processed focused refinement map of the upper Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Additional map: post-processed focused refinement map of the Ufd1/Npl4/polyubiquitin density...

Fileemd_27277_additional_7.map
Annotationpost-processed focused refinement map of the Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 1 of the Ufd1/Npl4/polyubiquitin...

Fileemd_27277_additional_8.map
Annotationfocused refinement half map 1 of the Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Additional map: focused refinement half map 2 of the Ufd1/Npl4/polyubiquitin...

Fileemd_27277_additional_9.map
Annotationfocused refinement half map 2 of the Ufd1/Npl4/polyubiquitin density of the ubiquitin unfolded state 'uC'
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Half map: overall refinement half map 2 of the ubiquitin unfolded state 'uC'

Fileemd_27277_half_map_1.map
Annotationoverall refinement half map 2 of the ubiquitin unfolded state 'uC'
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Half map: overall refinement half map 1 of the ubiquitin unfolded state 'uC'

Fileemd_27277_half_map_2.map
Annotationoverall refinement half map 1 of the ubiquitin unfolded state 'uC'
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Sample components

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

EntireName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
Components
  • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
    • Complex: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
      • 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 Ufd1/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 fol...

SupramoleculeName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
type: complex / ID: 1 / 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 substrate-bound complex engaged with two folded ubiquitin ...Details: 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 uC)
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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

SupramoleculeName: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two folded ubiquitin moieties and one unfolded ubiquitin in presence of SUMO-ubquitin(K48polyUb)-mEOS and ATP, state 2 (uC)
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Details: overall refinement map is "uncopenC_overall_390_postprocess_masked.mrc" and associated half maps are "uncopenC_overall_half1.mrc" and "uncopenC_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 / Parent: 1 / Macromolecule list: #1
Details: focused refinement map of the Cdc48 is "uncopenC_cdc48_533_postprocess_masked.mrc" and associated half maps are "uncopenC_cdc48_half1.mrc" and "uncopenC_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 / Parent: 1 / Macromolecule list: #2-#4
Details: Focused refinement map of the Ufd1/Npl4 tower with polyubiquitin is "uncopenC_tower_399_postprocess_masked.mrc" and associated half maps are "uncopenC_tower_half1.mrc" and "uncopenC_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 / Parent: 3 / Macromolecule list: #1
Details: focused refinement map of the D1/D2 ATPase domains of Cdc48 is "uncopenC_atpase_526_postprocess_masked.mrc" and associated half maps are "uncopenC_atpase_half1.mrc" and "uncopenC_atpase_half2.mrc"
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #6: Two folded ubiqutin moieties bound to Ufd1/Npl4

SupramoleculeName: Two folded ubiqutin moieties bound to Ufd1/Npl4 / type: complex / ID: 6 / Parent: 4 / Macromolecule list: #2, #4
Details: Focused refinement map of the Ufd1/Npl4 tower with polyubiquitin is "uncopenC_tower_399_postprocess_masked.mrc" and associated half maps are "uncopenC_tower_half1.mrc" and "uncopenC_tower_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

UniProtKB: Cell division control protein 48

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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

UniProtKB: Nuclear protein localization protein 4

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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

UniProtKB: Ubiquitin fusion degradation protein 1

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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: 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

UniProtKB: Polyubiquitin

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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: 48941
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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