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- EMDB-8659: VCP like ATPase from T. acidophilum (VAT) - Substrate bound confo... -

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Basic information

Entry
Database: EMDB / ID: EMD-8659
TitleVCP like ATPase from T. acidophilum (VAT) - Substrate bound conformation
Map dataVCP like ATPase from T. acidophilum - Substrate engaged state
Sample
  • Complex: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
    • Protein or peptide: VCP-like ATPase
KeywordsAAA+ / ATPase / Complex / unfoldase / HYDROLASE
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
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, 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
Biological speciesThermoplasma acidophilum (acidophilic) / Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165) (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsRipstein ZA / Huang R
Funding support Canada, 2 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)MOP-81294 Canada
Canadian Institutes of Health Research (CIHR)MOP-133408 Canada
CitationJournal: Elife / Year: 2017
Title: Structure of a AAA+ unfoldase in the process of unfolding substrate.
Authors: Zev A Ripstein / Rui Huang / Rafal Augustyniak / Lewis E Kay / John L Rubinstein /
Abstract: AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the homologue of eukaryotic CDC48/p97, works in ...AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.
History
DepositionMar 31, 2017-
Header (metadata) releaseApr 26, 2017-
Map releaseApr 26, 2017-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.137
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.137
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5vca
  • Surface level: 0.137
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8659.png

Downloads & links

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Map

FileDownload / File: emd_8659.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVCP like ATPase from T. acidophilum - Substrate engaged state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.137 / Movie #1: 0.137
Minimum - Maximum-0.41148973 - 0.7432196
Average (Standard dev.)-0.0001491764 (±0.027273158)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 371.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z371.200371.200371.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-36
NX/NY/NZ528549
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.4110.743-0.000

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Supplemental data

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Sample components

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Entire : Structural basis for substrate unfolding by the VCP like ATPase f...

EntireName: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
Components
  • Complex: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
    • Protein or peptide: VCP-like ATPase

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Supramolecule #1: Structural basis for substrate unfolding by the VCP like ATPase f...

SupramoleculeName: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Stacked-ring state, ATPgS loaded N-terminal domain removed
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 380 KDa

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Macromolecule #1: VCP-like ATPase

MacromoleculeName: VCP-like ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165) (acidophilic)
Strain: ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165
Molecular weightTheoretical: 62.996246 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEVSRISYED IGGLSEQLGK IREMIELPLK HPELFERLGI TPPKGVILYG PPGTGKTLIA RAVANESGAN FLSINGPEIM SKYYGQSEQ KLREIFSKAE ETAPSIIFID EIDSIAPKRE EVQGEVERRV VAQLLTLMDG MKERGHVIVI GATNRIDAID P ALRRPGRF ...String:
MEVSRISYED IGGLSEQLGK IREMIELPLK HPELFERLGI TPPKGVILYG PPGTGKTLIA RAVANESGAN FLSINGPEIM SKYYGQSEQ KLREIFSKAE ETAPSIIFID EIDSIAPKRE EVQGEVERRV VAQLLTLMDG MKERGHVIVI GATNRIDAID P ALRRPGRF DREIEIGVPD RNGRKEILMI HTRNMPLGMS EEEKNKFLEE MADYTYGFVG ADLAALVRES AMNALRRYLP EI DLDKPIP TEILEKMVVT EDDFKNALKS IEPSSLREVM VEVPNVHWDD IGGLEDVKRE IKETVELPLL KPDVFKRLGI RPS KGFLLY GPPGVGKTLL AKAVATESNA NFISIKGPEV LSKWVGESEK AIREIFKKAK QVAPAIVFLD EIDSIAPRRG TTSD SGVTE RIVNQLLTSL DGIEVMNGVV VIGATNRPDI MDPALLRAGR FDKLIYIPPP DKEARLSILK VHTKNMPLAP DVDLN DIAQ RTEGYVGADL ENLCREAGMN AYRENPDATS VSQKNFLDAL KTIRPSVDEE VIKFYRTLSE TMSKSVSERR KQLQDQ GLY L

UniProtKB: VCP-like ATPase

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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

Concentration20 mg/mL
BufferpH: 7.5
GridModel: Electron Microscopy Sciences M400 / Material: COPPER/RHODIUM / Mesh: 400 / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 3 / Number real images: 246 / Average exposure time: 15.0 sec. / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 171381
Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated by stochastic gradient descent algorithm in CryoSPARC
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.2) / Number images used: 75205
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.2)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 0.2) / Software - details: Ab intio with multiple classess

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Atomic model buiding 1

Initial modelPDB ID:

5vc7


Chain - Chain ID: A / Chain - Residue range: 183-726 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5vca:
VCP like ATPase from T. acidophilum (VAT)-Substrate bound conformation

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