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- EMDB-20000: Cdc48-Ufd1/Npl4 complex processing poly-ubiquitinated substrate i... -

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Basic information

Entry
Database: EMDB / ID: EMD-20000
TitleCdc48-Ufd1/Npl4 complex processing poly-ubiquitinated substrate in the presence of ADP-BeFx, state 2
Map dataMap sharpened with a B factor of -100
Sample
  • Complex: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ADP-BeFx, state 2
    • Protein or peptide: Cell division control protein 48
    • Protein or peptide: poly(alanine) substrate
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE ION
KeywordsATPase / ATPase complex / ubiquitin / quality control / MOTOR PROTEIN
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-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 / replisome / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / : / ribosome-associated ubiquitin-dependent protein catabolic process / retrograde protein transport, ER to cytosol / protein quality control for misfolded or incompletely synthesized proteins / autophagosome maturation / polyubiquitin modification-dependent protein binding / rescue of stalled ribosome / ATP metabolic process / : / Neutrophil degranulation / ubiquitin binding / macroautophagy / positive regulation of protein localization to nucleus / proteasome-mediated ubiquitin-dependent protein catabolic process / endoplasmic reticulum membrane / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
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 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. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division control protein 48
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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
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  • Atomic models: PDB-6oab
  • Surface level: 0.5
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6oab
  • Imaged by Jmol
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Structure viewerEM map:
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Supplemental images

emd_20000.png

Downloads & links

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Map

FileDownload / File: emd_20000.map.gz / Format: CCP4 / Size: 48.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened with a B factor of -100
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.0817745 - 2.613347
Average (Standard dev.)0.013759519 (±0.086522624)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 269.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z234234234
origin x/y/z0.0000.0000.000
length x/y/z269.100269.100269.100
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS234234234
D min/max/mean-1.0822.6130.014

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Supplemental data

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Additional map: Map sharpened with a B factor of -150

Fileemd_20000_additional_1.map
AnnotationMap sharpened with a B factor of -150
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map from refinement

Fileemd_20000_additional_2.map
AnnotationUnsharpened map from refinement
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map 2 from refinement

Fileemd_20000_half_map_1.map
AnnotationHalf map 2 from refinement
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map 1 from refinement

Fileemd_20000_half_map_2.map
AnnotationHalf map 1 from refinement
Projections & Slices
AxesZYX

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Histogram

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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 ADP-BeFx, state 2
Components
  • Complex: Cdc48-Npl4 complex processing poly-ubiquitinated substrate in the presence of ADP-BeFx, state 2
    • Protein or peptide: Cell division control protein 48
    • Protein or peptide: poly(alanine) substrate
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE 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 ADP-BeFx, state 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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: 5 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 92.106914 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 VFLDELDSIA 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: poly(alanine) substrate

MacromoleculeName: poly(alanine) substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.723883 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AAAAAAAAAA AAAAAAAAAA AAAA

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 10 / 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 #4: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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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 TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 44.7 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93395

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