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- EMDB-1193: Motions and negative cooperativity between p97 domains revealed b... -

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Basic information

Entry
Database: EMDB / ID: EMD-1193
TitleMotions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model.
Map dataAAA+ ATPase p97 in the presence of AMPPNP
Sample
  • Sample: Rat liver endogenous AAA ATPase p97
  • Protein or peptide: p97
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.0 Å
AuthorsBeuron F / Flynn TC / Ma J / Kondo H / Zhang X / Freemont PS
CitationJournal: J Mol Biol / Year: 2003
Title: Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model.
Authors: Fabienne Beuron / Terence C Flynn / Jianpeng Ma / Hisao Kondo / Xiaodong Zhang / Paul S Freemont /
Abstract: p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic ...p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function.
History
DepositionFeb 22, 2006-
Header (metadata) releaseFeb 23, 2006-
Map releaseFeb 23, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07076
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07076
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1193.gif

Downloads & links

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Map

FileDownload / File: emd_1193.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAAA+ ATPase p97 in the presence of AMPPNP
Voxel sizeX=Y=Z: 2.6 Å
Density
Contour Level1: 0.0593 / Movie #1: 0.07076
Minimum - Maximum-0.354279 - 0.508567
Average (Standard dev.)0.00033594 (±0.0294936)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-75-75-75
Dimensions150150150
Spacing150150150
CellA=B=C: 390 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z390.000390.000390.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-96-96-96
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS-75-75-75
NC/NR/NS150150150
D min/max/mean-0.3540.5090.000

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Supplemental data

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Sample components

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Entire : Rat liver endogenous AAA ATPase p97

EntireName: Rat liver endogenous AAA ATPase p97
Components
  • Sample: Rat liver endogenous AAA ATPase p97
  • Protein or peptide: p97

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Supramolecule #1000: Rat liver endogenous AAA ATPase p97

SupramoleculeName: Rat liver endogenous AAA ATPase p97 / type: sample / ID: 1000 / Oligomeric state: one homohexamer of p97 / Number unique components: 1
Molecular weightExperimental: 530 KDa / Theoretical: 580 KDa

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Macromolecule #1: p97

MacromoleculeName: p97 / type: protein_or_peptide / ID: 1 / Name.synonym: VCP / Number of copies: 6 / Oligomeric state: homohexamer / Recombinant expression: No
Source (natural)Organism: Rattus norvegicus (Norway rat) / Tissue: liver / Location in cell: cytoplasmic
Molecular weightExperimental: 97 MDa / Theoretical: 89 MDa

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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: 7.4 / Details: 20 mM TrisHCl, 150 mM KCl, 1 mM DTT pH 7.4
GridDetails: lacey carbon film
VitrificationCryogen name: ETHANE / Instrument: LEICA KF80 / Details: Vitrification instrument: Reichert KF 80 plunger / Method: blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
DateJul 1, 2000
Image recordingDigitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 15 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.4 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 38000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Detailsnumber of particles prior to symmetrisation
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic / Number images used: 3375
Final two d classificationNumber classes: 167

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