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

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Basic information

Entry
Database: PDB / ID: 5vca
TitleVCP like ATPase from T. acidophilum (VAT)-Substrate bound conformation
ComponentsVCP-like ATPase
KeywordsHYDROLASE / AAA+ / ATPase / Complex / unfoldase
Function / homology
Function and homology information


intracellular membrane-bounded organelle / ATP hydrolysis activity / ATP binding / cytoplasm
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsRipstein, Z.A. / Huang, R. / Augustyniak, R. / Kay, L.E. / Rubinstein, J.L.
Funding support Canada, 2items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2017Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Nov 6, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
P: VCP-like ATPase
M: VCP-like ATPase
N: VCP-like ATPase
O: VCP-like ATPase
Q: VCP-like ATPase
R: VCP-like ATPase


Theoretical massNumber of molelcules
Total (without water)377,9776
Polymers377,9776
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
VCP-like ATPase


Mass: 62996.246 Da / Num. of mol.: 6 / Fragment: UNP residues 183-745
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) 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
Gene: vat, Ta0840 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: O05209
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structural basis for substrate unfolding by the VCP like ATPase from T. Acidophilum
Type: COMPLEX
Details: Stacked-ring state, ATPgS loaded N-terminal domain removed
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: PPROEX
Buffer solutionpH: 7.5
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Electron Microscopy Sciences M400
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot for 4 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 25000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1700 nm / Cs: 2 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 246
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4CTF correction
7UCSF Chimera1.1model fitting
8NAMD2model fitting
10cryoSPARC0.2initial Euler assignment
11cryoSPARC0.2final Euler assignment
12cryoSPARC0.2classificationAb intio with multiple classess
13cryoSPARC0.23D reconstruction
20PHENIX1.10.1-2155model refinementReal space refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 171381
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75205 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5VC7

5vc7


Pdb chain-ID: A / Accession code: 5VC7 / Pdb chain residue range: 183-726 / Source name: PDB / Type: experimental model

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