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- PDB-5g4g: Structure of the ATPgS-bound VAT complex -

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Basic information

Entry
Database: PDB / ID: 5g4g
TitleStructure of the ATPgS-bound VAT complex
ComponentsVCP-LIKE ATPASE
KeywordsHYDROLASE / VAT / PROTEASOME / PROTEIN DYNAMICS / UNFOLDASE / CONFORMATIONS
Function / homology
Function and homology information


hydrolase 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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / 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 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / 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: 7.8 Å
AuthorsHuang, R. / Ripstein, Z.A. / Augustyniak, R. / Lazniewski, M. / Ginalski, K. / Kay, L.E. / Rubinstein, J.L.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study.
Authors: Rui Huang / Zev A Ripstein / Rafal Augustyniak / Michal Lazniewski / Krzysztof Ginalski / Lewis E Kay / John L Rubinstein /
Abstract: The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase ...The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.
History
DepositionMay 12, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 27, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2016Group: Database references
Revision 1.2Oct 3, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • EMDB-3435
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Assembly

Deposited unit
A: VCP-LIKE ATPASE
B: VCP-LIKE ATPASE
C: VCP-LIKE ATPASE
D: VCP-LIKE ATPASE
E: VCP-LIKE ATPASE
F: VCP-LIKE ATPASE


Theoretical massNumber of molelcules
Total (without water)482,5536
Polymers482,5536
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
VCP-LIKE ATPASE


Mass: 80425.570 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) THERMOPLASMA ACIDOPHILUM (acidophilic) / Plasmid: PPROEX / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: O05209

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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: VAT (CDC48 HOMOLOGUE) / Type: COMPLEX
Buffer solutionName: 50 MM HEPES, 100 MM NACL, 5MM ATPGS / pH: 7.5 / Details: 50 MM HEPES, 100 MM NACL, 5MM ATPGS
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE-PROPANE MIXTURE, HUMIDITY- 100, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT FOR 4 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: May 1, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 25000 X / Calibrated magnification: 34483 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1800 nm / Cs: 2 mm
Image recordingElectron dose: 32 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansNum. digital images: 514

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Processing

EM softwareName: RELION / Category: 3D reconstruction
CTF correctionDetails: EACH MICROGRAPH
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 7.8 Å / Num. of particles: 16538 / Nominal pixel size: 1.45 Å / Actual pixel size: 1.45 Å / Magnification calibration: CRYSTAL LATTICE MEASURMENT
Details: HOMOLOGY MODEL WAS FLEXIBLY FIT INTO DENSITY THEN STEREO- CHEMICALLY REFINED SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3435. (DEPOSITION ID: 14516).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--MOLECULAR DYNAMICS FLEXIBLE FITTING REFINEMENT PROTOCOL--EM
RefinementHighest resolution: 7.8 Å
Refinement stepCycle: LAST / Highest resolution: 7.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms33462 0 0 0 33462

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