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- EMDB-8658: VCP like ATPase from T. acidophilum (VAT) - conformation 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-8658
TitleVCP like ATPase from T. acidophilum (VAT) - conformation 1
Map dataVAT hexamer in stacked-ring conformation
Sample
  • Complex: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
    • Protein or peptide: VCP-like ATPase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Function / homology
Function and homology information


macromolecule metabolic process / primary metabolic process / : / response to stimulus / 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, 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
Biological speciesThermoplasma acidophilum (acidophilic) / Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165) (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsRipstein ZA / Huang R / Augustyniak R / Kay LE / Rubinstein JL
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-
UpdateJan 15, 2020-
Current statusJan 15, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5vc7
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8658.png

Downloads & links

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Map

FileDownload / File: emd_8658.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVAT hexamer in stacked-ring conformation
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.6932421 - 1.1776209
Average (Standard dev.)-0.0007511465 (±0.041298486)
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-0.6931.178-0.001

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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
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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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: #1
Details: Stacked-ring state, ATPgS loaded N-terminal domain removed
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: PPROEX
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

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 12 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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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: 30.0 nm
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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

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Image processing

Particle selectionNumber selected: 171381
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated by stochastic gradient descent algorithm in CryoSPARC
Initial 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
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.2) / Number images used: 75205

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5vc7:
VCP like ATPase from T. acidophilum (VAT) - conformation 1

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