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- PDB-8das: Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubi... -

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Basic information

Entry
Database: PDB / ID: 8das
TitleSaccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)
Components
  • Cell division control protein 48
  • Nuclear protein localization protein 4
  • Ubiquitin fusion degradation protein 1
  • Ubiquitin
KeywordsMOTOR PROTEIN / ATPASE / ATPASE COMPLEX / UBIQUITIN / SUMO / SMT3 / QUALITY CONTROL
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 / ribosomal large subunit export from nucleus / mRNA transport / rescue of stalled ribosome / : / ATP metabolic process / Neutrophil degranulation / ubiquitin binding / macroautophagy / modification-dependent protein catabolic process / ribosomal large subunit assembly / protein tag activity / positive regulation of protein localization to nucleus / ribosome biogenesis / cytoplasmic translation / cytosolic large ribosomal subunit / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / nuclear membrane / 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 / 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 / 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 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLee, H.G. / Lima, C.D.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cell division control protein 48
B: Cell division control protein 48
C: Cell division control protein 48
D: Cell division control protein 48
E: Cell division control protein 48
F: Cell division control protein 48
G: Nuclear protein localization protein 4
H: Ubiquitin fusion degradation protein 1
J: Ubiquitin
K: Ubiquitin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)683,27624
Polymers677,61810
Non-polymers5,65714
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 10 molecules ABCDEFGHJK

#1: Protein
Cell division control protein 48 / Cell division cycle protein 48 / Transitional endoplasmic reticulum ATPase homolog


Mass: 92389.195 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CDC48, YDL126C / Production host: Escherichia coli (E. coli) / References: UniProt: P25694, vesicle-fusing ATPase
#2: Protein Nuclear protein localization protein 4 / HMG-CoA reductase degradation protein 4


Mass: 66144.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NPL4, HRD4, YBR170C, YBR1231 / Production host: Escherichia coli (E. coli) / References: UniProt: P33755
#3: Protein Ubiquitin fusion degradation protein 1 / UB fusion protein 1 / Polymerase-interacting protein 3


Mass: 40001.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: UFD1, PIP3, YGR048W / Production host: Escherichia coli (E. coli) / References: UniProt: P53044
#4: Protein Ubiquitin /


Mass: 8568.769 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
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
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RPL40A, UBI1, YIL148W / Production host: Escherichia coli (E. coli) / References: UniProt: P0CH08

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Non-polymers , 3 types, 14 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)COMPLEXUfd1/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)#1-#40RECOMBINANT
2Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex bound to two ubiquitin moieties in presence of SUMO-ubiquitin(K48polyUb)-mEOS and ATP, state 1 (intA)COMPLEXoverall refinement map is "uncclosedE_overall_291_postprocess_masked.mrc" from half maps "uncclosedE_overall_half1.mrc" and "uncclosedE_overall_half2.mrc"#1-#41RECOMBINANT
3Cdc48 hexamerCOMPLEXfocused 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"#11RECOMBINANT
4Two folded ubiquitin moieties bound to the Ufd1/Npl4 towerCOMPLEXfocused 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"#2-#41RECOMBINANT
5D1/D2 ATPase domains of the Cdc48 hexamerCOMPLEXfocused 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"#13RECOMBINANT
6Two folded ubiqutin moieties bound to Npl4COMPLEXfocused 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"#2, #44RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Saccharomyces cerevisiae (brewer's yeast)4932
32Saccharomyces cerevisiae (brewer's yeast)4932
43Saccharomyces cerevisiae (brewer's yeast)4932
54Saccharomyces cerevisiae (brewer's yeast)4932
65Saccharomyces cerevisiae (brewer's yeast)4932
76Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
65Escherichia coli (E. coli)562
76Escherichia coli (E. coli)562
Buffer solutionpH: 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.
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Ufd1/Npl4/Cdc48 was pre-incubated with SUMO-ubiquitin(K48polyUb)-mEOS and ATP
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 8 s wait, 4 s blot before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 70.577 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29595 / Symmetry type: POINT

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