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- EMDB-7478: Cdc48 in the presence of ATP-gamma-S -

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Basic information

Entry
Database: EMDB / ID: EMD-7478
TitleCdc48 in the presence of ATP-gamma-S
Map dataCdc48-Npl4 complex in the presence of ATP-gamma-S
Sample
  • Organelle or cellular component: Cdc48
    • Protein or peptide: Cdc48
Biological speciesChaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsKim KH / Bodnar NO
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4.
Authors: Nicholas O Bodnar / Kelly H Kim / Zhejian Ji / Thomas E Wales / Vladimir Svetlov / Evgeny Nudler / John R Engen / Thomas Walz / Tom A Rapoport /
Abstract: Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains ...Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn-finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48's central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.
History
DepositionFeb 23, 2018-
Header (metadata) releaseMar 21, 2018-
Map releaseJul 4, 2018-
UpdateDec 5, 2018-
Current statusDec 5, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7478.png

Downloads & links

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Map

FileDownload / File: emd_7478.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCdc48-Npl4 complex in the presence of ATP-gamma-S
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 200 pix.
= 248. Å		1.24 Å/pix.
x 200 pix.
= 248. Å		1.24 Å/pix.
x 200 pix.
= 248. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.24 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.11144812 - 0.17007945
Average (Standard dev.)0.001122055 (±0.008527964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 248.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z248.000248.000248.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1110.1700.001

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Supplemental data

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Sample components

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Entire : Cdc48

EntireName: Cdc48
Components
  • Organelle or cellular component: Cdc48
    • Protein or peptide: Cdc48

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Supramolecule #1: Cdc48

SupramoleculeName: Cdc48 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Cdc48

MacromoleculeName: Cdc48 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAPESKDHKK KVNLTDPSGA EHREENDTAT AILKKKKKPN QLMVTDAVND DNSIIALSNN TMEALQLFRG DTVLVRGKKR RDTVLIVLAD DDLDDGSARI NRVVRHNLRV KHGDMITIHP CPDIKYAKRI AVLPIADTVE GLTGSLFDVF LAPYFREAYR PVRQGDLFTV ...String:
MAPESKDHKK KVNLTDPSGA EHREENDTAT AILKKKKKPN QLMVTDAVND DNSIIALSNN TMEALQLFRG DTVLVRGKKR RDTVLIVLAD DDLDDGSARI NRVVRHNLRV KHGDMITIHP CPDIKYAKRI AVLPIADTVE GLTGSLFDVF LAPYFREAYR PVRQGDLFTV RGGMRQVEFK VVEVDPPEYG IVAQDTIIHC EGEPIPREEE ENNLNEVGYD DIGGCRKQLA QIREMVELPL RHPQLFKSIG IKPPRGVLLY GPPGTGKTLM ARAVANETGA FFFLINGPEI MSKMAGESES NLRKAFEEAE KNSPAIIFID EIDSIAPKRE KTNGEVERRV VSQLLTLMDG MKARSNVVVM AATNRPNSID PALRRFGRFD REVDIGIPDP TGRLEILQIH TKNMKLADDV DLEQIAAETH GYVGSDLAAL CSEAAMQQIR EKMDLIDLDE DTIDAEVLDS LGVTMDNFRY ALGVSNPSAL REVAVVEVPN VRWEDIGGLE QVKQELKEQV QYPVDHPEKF LKFGLSPSRG VLFYGPPGTG KTMLAKAVAN ECAANFISVK GPELLSMWFG ESESNIRDIF DKARAAAPCV VFLDELDSIA KARGGSIGDA GGASDRVVNQ LLTEMDGMTS KKNVFVIGAT NRPEQLDPAL CRPGRLDQLI YVPLPDEAGR LSILKAQLRK TPVSKDVDLA YIASKTHGFS GADLAFITQR AVKLAIKESI AAEIERQKAR EAAGEDVNME DDEDPVPELT KRHFEEAMRD ARRSVSDVEI RRYEAFAQQM KNAGPGAFFK FPDSTTDNSA SNAAGNSFGD AGNDDDLYT

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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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES
150.0 mMsodium chlorideNaCl
5.0 mMmagnesium chlorideMgCl2
0.5 mMTCEP
100.0 uMATP-gamma-S
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 569 / Average exposure time: 6.0 sec. / Average electron dose: 1.04 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 29313
CTF correctionSoftware - Name: CTFFIND
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 3021
Initial angle assignmentType: COMMON LINE / Software - Name: SPARX
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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