+Open data
-Basic information
Entry | Database: PDB / ID: 5g4g | ||||||
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Title | Structure of the ATPgS-bound VAT complex | ||||||
Components | VCP-LIKE ATPASE | ||||||
Keywords | HYDROLASE / VAT / PROTEASOME / PROTEIN DYNAMICS / UNFOLDASE / CONFORMATIONS | ||||||
Function / homology | Function and homology information macromolecule metabolic process / primary metabolic process / response to stimulus / : / ATP hydrolysis activity / ATP binding Similarity search - Function | ||||||
Biological species | THERMOPLASMA ACIDOPHILUM (acidophilic) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.8 Å | ||||||
Authors | Huang, R. / Ripstein, Z.A. / Augustyniak, R. / Lazniewski, M. / Ginalski, K. / Kay, L.E. / Rubinstein, J.L. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5g4g.cif.gz | 688.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5g4g.ent.gz | 573.3 KB | Display | PDB format |
PDBx/mmJSON format | 5g4g.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g4/5g4g ftp://data.pdbj.org/pub/pdb/validation_reports/g4/5g4g | HTTPS FTP |
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-Related structure data
Related structure data | 3435MC 3436C 5g4fC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: VAT (CDC48 HOMOLOGUE) / Type: COMPLEX |
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Buffer solution | Name: 50 MM HEPES, 100 MM NACL, 5MM ATPGS / pH: 7.5 / Details: 50 MM HEPES, 100 MM NACL, 5MM ATPGS |
Specimen | Conc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: HOLEY CARBON |
Vitrification | Instrument: 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, |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 / Date: May 1, 2015 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 recording | Electron dose: 32 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
Image scans | Num. digital images: 514 |
-Processing
EM software | Name: RELION / Category: 3D reconstruction | ||||||||||||
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CTF correction | Details: EACH MICROGRAPH | ||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: FLEXIBLE FIT / Space: REAL Details: METHOD--MOLECULAR DYNAMICS FLEXIBLE FITTING REFINEMENT PROTOCOL--EM | ||||||||||||
Refinement | Highest resolution: 7.8 Å | ||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 7.8 Å
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