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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3435 | |||||||||
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Title | Structure of the ATPgS-bound VAT complex | |||||||||
![]() | Stacked-ring state | |||||||||
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![]() | VAT / proteasome / protein dynamics / unfoldase / conformations | |||||||||
Function / homology | ![]() metabolic process / retrograde protein transport, ER to cytosol / polyubiquitin modification-dependent protein binding / hydrolase activity / nucleotide binding / ATP hydrolysis activity / ATP binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.8 Å | |||||||||
![]() | Huang R / Ripstein ZA / Augustyniak R / Lazniewski M / Ginalski K / Kay LE / Rubinstein JL | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 5.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.5 KB 10.5 KB | Display Display | ![]() |
Images | ![]() | 96.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5g4gMC ![]() 3436C ![]() 5g4fC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Stacked-ring state | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.45 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : VAT (CDC48 homologue)
Entire | Name: VAT (CDC48 homologue) |
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Components |
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-Supramolecule #1000: VAT (CDC48 homologue)
Supramolecule | Name: VAT (CDC48 homologue) / type: sample / ID: 1000 / Oligomeric state: homohexamer / Number unique components: 1 |
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Molecular weight | Theoretical: 500 KDa |
-Macromolecule #1: VCP like ATPase from T. Acidophilum
Macromolecule | Name: VCP like ATPase from T. Acidophilum / type: protein_or_peptide / ID: 1 / Name.synonym: VAT / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 83 MDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: VCP-like ATPase GO: metabolic process, nucleotide binding, ATP binding, hydrolase activity InterPro: AAA+ ATPase domain, AAA ATPase, CDC48 family, Aspartate decarboxylase-like domain superfamily, ATPase, AAA-type, core, ATPase, AAA-type, conserved site, CDC48, domain 2, CDC48 domain 2-like ...InterPro: AAA+ ATPase domain, AAA ATPase, CDC48 family, Aspartate decarboxylase-like domain superfamily, ATPase, AAA-type, core, ATPase, AAA-type, conserved site, CDC48, domain 2, CDC48 domain 2-like superfamily, CDC48, N-terminal subdomain, P-loop containing nucleoside triphosphate hydrolase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 7 mg/mL |
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Buffer | pH: 7.5 / Details: 50 mM HEPES, 100 mM NaCl, 5mM ATPgS |
Grid | Details: 400 mesh Cu/Rh grid with homemade nanofabricated holy carbon support |
Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: Blot for 4 seconds before plunging |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Date | May 1, 2015 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 1.45 µm / Number real images: 514 / Average electron dose: 30 e/Å2 Details: Every image is the average of 30 frames recorded by the direct electron detector Bits/pixel: 32 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 25000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | Whole frame alignment was performed with alignframes_lmbfgs, and the resulting averages of frames were used for CTF determination with CTFFIND4. Automated particle picking was done in Relion. Individual particle alignment and exposure weighting was done with alignparts_lmbfgs. Classification and refinement were perfomed with Relion. |
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CTF correction | Details: Each Micrograph |
Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 16538 |