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- EMDB-0665: Cdc48-Ufd1/Npl4 complex processing poly-ubiquitinated substrate i... -

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Basic information

Entry
Database: EMDB / ID: EMD-0665
TitleCdc48-Ufd1/Npl4 complex processing poly-ubiquitinated substrate in the presence of ATP
Map dataComposite map, sharpened with a B factor of -150
Sample
  • Complex: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ATP
    • Protein or peptide: Cell division control protein 48
    • Protein or peptide: Ubiquitin
    • Protein or peptide: Nuclear protein localization protein 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
KeywordsATPase / ATPase complex / ubiquitin / quality control / MOTOR PROTEIN
Function / homology
Function and homology information


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 / sister chromatid biorientation / protein localization to vacuole / Hrd1p ubiquitin ligase ERAD-L complex / endoplasmic reticulum membrane fusion ...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 / sister chromatid biorientation / protein localization to vacuole / Hrd1p ubiquitin ligase ERAD-L complex / endoplasmic reticulum membrane fusion / ribophagy / DNA replication termination / RQC complex / 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 / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / endosome to plasma membrane protein transport / protein phosphatase regulator activity / Translesion Synthesis by POLH / piecemeal microautophagy of the nucleus / mating projection tip / 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 / nuclear outer membrane-endoplasmic reticulum membrane network / retrograde protein transport, ER to cytosol / nonfunctional rRNA decay / KEAP1-NFE2L2 pathway / Neddylation / protein quality control for misfolded or incompletely synthesized proteins / ribosomal large subunit export from nucleus / polyubiquitin modification-dependent protein binding / autophagosome maturation / mRNA transport / 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 / ribosome biogenesis / ribosomal large subunit assembly / ubiquitin-dependent protein catabolic process / nuclear membrane / cytosolic large ribosomal subunit / proteasome-mediated ubiquitin-dependent protein catabolic process / 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
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 ...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 / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / MPN domain / MPN domain profile. / AAA ATPase, AAA+ lid domain / AAA+ lid domain / 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
Ubiquitin-ribosomal protein eL40A fusion protein / Cell division control protein 48 / Nuclear protein localization protein 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsTwomey EC / Ji Z
Funding support United States, 3 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM052586 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM007753 United States
CitationJournal: Science / Year: 2019
Title: Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding.
Authors: Edward C Twomey / Zhejian Ji / Thomas E Wales / Nicholas O Bodnar / Scott B Ficarro / Jarrod A Marto / John R Engen / Tom A Rapoport /
Abstract: The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for ...The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.
History
DepositionMar 15, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oa9
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0665.png

Downloads & links

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Map

FileDownload / File: emd_0665.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map, sharpened with a B factor of -150
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-1.4414015 - 3.2028573
Average (Standard dev.)0.016643317 (±0.11144938)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-1.4413.2030.017

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Supplemental data

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Additional map: Local map for D1/Npl4 sharpened with a B factor of -100

Fileemd_0665_additional_1.map
AnnotationLocal map for D1/Npl4 sharpened with a B factor of -100
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Additional map: Full unsharpened map from refinement

Fileemd_0665_additional_2.map
AnnotationFull unsharpened map from refinement
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Additional map: Local map for D1/Npl4 sharpened with a B factor of -150

Fileemd_0665_additional_3.map
AnnotationLocal map for D1/Npl4 sharpened with a B factor of -150
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Additional map: Local map for D1/Npl4 sharpened with a B factor of -200

Fileemd_0665_additional_4.map
AnnotationLocal map for D1/Npl4 sharpened with a B factor of -200
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Additional map: Local map for D2 sharpened with a B factor of -100

Fileemd_0665_additional_5.map
AnnotationLocal map for D2 sharpened with a B factor of -100
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Additional map: Local map for D2 sharpened with a B factor of -150

Fileemd_0665_additional_6.map
AnnotationLocal map for D2 sharpened with a B factor of -150
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Additional map: Local map for D2 sharpened with a B factor of -200

Fileemd_0665_additional_7.map
AnnotationLocal map for D2 sharpened with a B factor of -200
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Half map: Half map 1

Fileemd_0665_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_0665_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the...

EntireName: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ATP
Components
  • Complex: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ATP
    • Protein or peptide: Cell division control protein 48
    • Protein or peptide: Ubiquitin
    • Protein or peptide: Nuclear protein localization protein 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the...

SupramoleculeName: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ATP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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: 92.105922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGEEHKPLLD ASGVDPREED KTATAILRRK KKDNMLLVDD AINDDNSVIA INSNTMDKLE LFRGDTVLVK GKKRKDTVLI VLIDDELED GACRINRVVR NNLRIRLGDL VTIHPCPDIK YATRISVLPI ADTIEGITGN LFDVFLKPYF VEAYRPVRKG D HFVVRGGM ...String:
MGEEHKPLLD ASGVDPREED KTATAILRRK KKDNMLLVDD AINDDNSVIA INSNTMDKLE LFRGDTVLVK GKKRKDTVLI VLIDDELED GACRINRVVR NNLRIRLGDL VTIHPCPDIK YATRISVLPI ADTIEGITGN LFDVFLKPYF VEAYRPVRKG D HFVVRGGM RQVEFKVVDV EPEEYAVVAQ DTIIHWEGEP INREDEENNM NEVGYDDIGG CRKQMAQIRE MVELPLRHPQ LF KAIGIKP PRGVLMYGPP GTGKTLMARA VANETGAFFF LINGPEVMSK MAGESESNLR KAFEEAEKNA PAIIFIDEID SIA PKRDKT NGEVERRVVS QLLTLMDGMK ARSNVVVIAA TNRPNSIDPA LRRFGRFDRE VDIGIPDATG RLEVLRIHTK NMKL ADDVD LEALAAETHG YVGADIASLC SEAAMQQIRE KMDLIDLDED EIDAEVLDSL GVTMDNFRFA LGNSNPSALR ETVVE SVNV TWDDVGGLDE IKEELKETVE YPVLHPDQYT KFGLSPSKGV LFYGPPGTGK TLLAKAVATE VSANFISVKG PELLSM WYG ESESNIRDIF DKARAAAPTV VFLDQLDSIA KARGGSLGDA GGASDRVVNQ LLTEMDGMNA KKNVFVIGAT NRPDQID PA ILRPGRLDQL IYVPLPDENA RLSILNAQLR KTPLEPGLEL TAIAKATQGF SGADLLYIVQ RAAKYAIKDS IEAHRQHE A EKEVKVEGED VEMTDEGAKA EQEPEVDPVP YITKEHFAEA MKTAKRSVSD AELRRYEAYS QQMKASRGQF SNFNFNDAP LGTTATDNAN SNNSAPSGAG AAFGSNAEED DDLYS

UniProtKB: Cell division control protein 48

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Macromolecule #2: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 2 / 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: Ubiquitin-ribosomal protein eL40A fusion protein

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Macromolecule #3: Nuclear protein localization protein 4

MacromoleculeName: Nuclear protein localization protein 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 65.862062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLIRFRSKNG THRVSCQEND LFGTVIEKLV GNLDPNADVD TFTVCEKPGQ GIHAVSELAD RTVMDLGLKH GDMLILNYSD KPANEKDGV NVEIGSVGID SKGIRQHRYG PLRIKELAVD EELEKEDGLI PRQKSKLCKH GDRGMCEYCS PLPPWDKEYH E KNKIKHIS ...String:
MLIRFRSKNG THRVSCQEND LFGTVIEKLV GNLDPNADVD TFTVCEKPGQ GIHAVSELAD RTVMDLGLKH GDMLILNYSD KPANEKDGV NVEIGSVGID SKGIRQHRYG PLRIKELAVD EELEKEDGLI PRQKSKLCKH GDRGMCEYCS PLPPWDKEYH E KNKIKHIS FHSYLKKLNE NANKKENGSS YISPLSEPDF RINKRCHNGH EPWPRGICSK CQPSAITLQQ QEFRMVDHVE FQ KSEIINE FIQAWRYTGM QRFGYMYGSY SKYDNTPLGI KAVVEAIYEP PQHDEQDGLT MDVEQVKNEM LQIDRQAQEM GLS RIGLIF TDLSDAGAGD GSVFCKRHKD SFFLSSLEVI MAARHQTRHP NVSKYSEQGF FSSKFVTCVI SGNLEGEIDI SSYQ VSTEA EALVTADMIS GSTFPSMAYI NDTTDERYVP EIFYMKSNEY GITVKENAKP AFPVDYLLVT LTHGFPNTDT ETNSK FVSS TGFPWSNRQA MGQSQDYQEL KKYLFNVASS GDFNLLHEKI SNFHLLLYIN SLQILSPDEW KLLIESAVKN EWEESL LKL VSSAGWQTLV MILQESG

UniProtKB: Nuclear protein localization protein 4

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 9 / 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 #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / 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 #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / 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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: GATAN CRYOPLUNGE 3
Details: Waited 20 seconds before blotting for 2.5-3 seconds..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 54.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6chs

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 127261
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)

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