+Open data
-Basic information
Entry | Database: PDB / ID: 8u0v | ||||||||||||
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Title | S. cerevisiae Pex1/Pex6 with 1 mM ATP | ||||||||||||
Components |
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Keywords | MOTOR PROTEIN / Peroxisome AAA-ATPase unfoldase | ||||||||||||
Function / homology | Function and homology information protein import into peroxisome matrix, receptor recycling / protein import into peroxisome matrix / protein transporter activity / peroxisomal membrane / ATPase complex / protein unfolding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / peroxisome / ATP hydrolysis activity / ATP binding / cytosol Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.89 Å | ||||||||||||
Authors | Gardner, B.M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: J Biol Chem / Year: 2024 Title: The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1. Authors: Bashir A Ali / Ryan M Judy / Saikat Chowdhury / Nicole K Jacobsen / Dominic T Castanzo / Kaili L Carr / Chris D Richardson / Gabriel C Lander / Andreas Martin / Brooke M Gardner / Abstract: The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, ...The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8u0v.cif.gz | 2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8u0v.ent.gz | 1.4 MB | Display | PDB format |
PDBx/mmJSON format | 8u0v.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8u0v_validation.pdf.gz | 2.2 MB | Display | wwPDB validaton report |
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Full document | 8u0v_full_validation.pdf.gz | 2.5 MB | Display | |
Data in XML | 8u0v_validation.xml.gz | 178.5 KB | Display | |
Data in CIF | 8u0v_validation.cif.gz | 256.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u0/8u0v ftp://data.pdbj.org/pub/pdb/validation_reports/u0/8u0v | HTTPS FTP |
-Related structure data
Related structure data | 41788MC 8u0xC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 118653.977 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PEX1, PAS1, YKL197C / Production host: Escherichia coli (E. coli) References: UniProt: P24004, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement #2: Protein | Mass: 117306.758 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PEX6, PAS8, YNL329C, N0310 / Production host: Escherichia coli (E. coli) References: UniProt: P33760, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement #3: Chemical | ChemComp-ATP / Has ligand of interest | Y | |
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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: Pex1/Pex6 AAA-ATPase / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.637 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 43478 X / Calibrated magnification: 43478 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Temperature (max): 83 K / Temperature (min): 83 K |
Image recording | Average exposure time: 12 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2355327 Details: Lowpass (20A) template picking of 200nm diameter particles using cryoSPARC 3.3.2. | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106005 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | ||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 246.24 Å2 | ||||||||||||||||||||||||||||
Refine LS restraints |
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