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- EMDB-2038: Mammalian AAA ATPase p97 A232E mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-2038
TitleMammalian AAA ATPase p97 A232E mutant
Map datap97 A232E
Sample
  • Sample: Full length p97 IBMPFD mutant A232E
  • Protein or peptide: p97
Keywordsp97 / VCP / IBMPFD / A232E
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 Å
AuthorsNiwa H / Ewens CA / Tsang C / Yeung HO / Zhang X / Freemont PS
CitationJournal: J Biol Chem / Year: 2012
Title: The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.
Authors: Hajime Niwa / Caroline A Ewens / Chun Tsang / Heidi O Yeung / Xiaodong Zhang / Paul S Freemont /
Abstract: p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase ...p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.
History
DepositionJan 21, 2012-
Header (metadata) releaseFeb 2, 2012-
Map releaseFeb 15, 2012-
UpdateFeb 15, 2012-
Current statusFeb 15, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 13.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 13.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2038.tif

Downloads & links

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Map

FileDownload / File: emd_2038.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationp97 A232E
Voxel sizeX=Y=Z: 3.52 Å
Density
Contour LevelBy AUTHOR: 13.800000000000001 / Movie #1: 13.8
Minimum - Maximum0.0 - 141.231552120000003
Average (Standard dev.)0.63924396 (±6.21090364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.523.523.52
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z450.560450.560450.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.000141.2320.639

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Supplemental data

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Sample components

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Entire : Full length p97 IBMPFD mutant A232E

EntireName: Full length p97 IBMPFD mutant A232E
Components
  • Sample: Full length p97 IBMPFD mutant A232E
  • Protein or peptide: p97

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Supramolecule #1000: Full length p97 IBMPFD mutant A232E

SupramoleculeName: Full length p97 IBMPFD mutant A232E / type: sample / ID: 1000 / Oligomeric state: one p97 homohexamer / Number unique components: 1
Molecular weightExperimental: 540 KDa / Theoretical: 540 KDa

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Macromolecule #1: p97

MacromoleculeName: p97 / type: protein_or_peptide / ID: 1 / Name.synonym: p97 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 250 mM KCl, 25 mM HEPES
GridDetails: holey carbon film on copper
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingNumber real images: 50 / Average electron dose: 10 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.005 µm / Nominal defocus min: 0.002 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Particles
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: imagic / Number images used: 1029

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