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- EMDB-3436: Structure of the ADP-bound VAT complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3436
TitleStructure of the ADP-bound VAT complex
Map dataADP-bound VAT complex
Sample
  • Sample: VAT (CDC48 homologue)
  • Protein or peptide: VCP like ATPase from T. Acidophilum
KeywordsVAT / proteasome / protein dynamics / unfoldase / conformations / AAA+ ATPase
Function / homology
Function and homology information


metabolic process / hydrolase activity / nucleotide binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
AAA ATPase, CDC48 family / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 ...AAA ATPase, CDC48 family / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / CDC48 domain 2-like superfamily / : / Aspartate decarboxylase-like domain superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsHuang R / Ripstein ZA / Augustyniak R / Lazniewski M / Ginalski K / Kay LE / Rubinstein JL
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 11, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseJul 20, 2016-
UpdateAug 24, 2016-
Current statusAug 24, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5g4f
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

wtVAT_ADP2.png

Downloads & links

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Map

FileDownload / File: emd_3436.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationADP-bound VAT complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.10781565 - 0.26468402
Average (Standard dev.)0.00088653 (±0.00825485)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1080.2650.001

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Supplemental data

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Sample components

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Entire : VAT (CDC48 homologue)

EntireName: VAT (CDC48 homologue)
Components
  • Sample: VAT (CDC48 homologue)
  • Protein or peptide: VCP like ATPase from T. Acidophilum

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Supramolecule #1000: VAT (CDC48 homologue)

SupramoleculeName: VAT (CDC48 homologue) / type: sample / ID: 1000 / Oligomeric state: homohexamer / Number unique components: 1
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: VCP like ATPase from T. Acidophilum

MacromoleculeName: 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
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 830 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pProEx
SequenceUniProtKB: 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

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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

Concentration7 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES, 100 mM NaCl, 5mM ADP
GridDetails: 400 mesh Cu/Rh grid with homemade nanofabricated holy carbon support
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Details: Sample held at 4 degrees Celsius before freezing / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 175,000 times magnification by inspection of FFT
DateMay 1, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 1.45 µm / Number real images: 912 / Average electron dose: 32 e/Å2
Details: Every image is the average of 30 frames recorded by the direct electron detector
Bits/pixel: 32
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsWhole frame alignment was performed with alignframes_lmbfgs, and the resulting averages of frames were used for CTF parameter determination with CTFFIND4. Anisotropic magnification was corrected for particle images and CTF parameters. 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.
CTF correctionDetails: Each Micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 71258

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