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- PDB-8u0v: S. cerevisiae Pex1/Pex6 with 1 mM ATP -

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Basic information

Entry
Database: PDB / ID: 8u0v
TitleS. cerevisiae Pex1/Pex6 with 1 mM ATP
Components
  • Peroxisomal ATPase PEX1
  • Peroxisomal ATPase PEX6
KeywordsMOTOR 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
Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like 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 ...Peroxisome biogenesis factor 1, N-terminal, psi beta-barrel fold / : / Peroxisome biogenesis factor 1, N-terminal / CDC48 domain 2-like 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
ADENOSINE-5'-TRIPHOSPHATE / Peroxisomal ATPase PEX1 / Peroxisomal ATPase PEX6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.89 Å
AuthorsGardner, B.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)K99GM121880 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS095892 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: 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
DepositionAug 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Peroxisomal ATPase PEX1
C: Peroxisomal ATPase PEX1
E: Peroxisomal ATPase PEX1
F: Peroxisomal ATPase PEX6
B: Peroxisomal ATPase PEX6
D: Peroxisomal ATPase PEX6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)712,95416
Polymers707,8826
Non-polymers5,07210
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Peroxisomal ATPase PEX1 / Peroxin-1 / Peroxisomal assembly protein 1 / Peroxisome biogenesis protein PAS1


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 Peroxisomal ATPase PEX6 / Peroxin-6 / Peroxisomal assembly protein 8


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 / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY

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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: Pex1/Pex6 AAA-ATPase / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.637 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
11 mMATPAdenosine triphosphate1
260 mMHEPES1
350 mMsodium chlorideNaClSodium chloride1
450 mMpotassium chlorideKCl1
510 mMmagnesium chlorideMgCl21
65 %glycerol1
70.5 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderTemperature (max): 83 K / Temperature (min): 83 K
Image recordingAverage exposure time: 12 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7PHENIXmodel fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2355327
Details: Lowpass (20A) template picking of 200nm diameter particles using cryoSPARC 3.3.2.
3D reconstructionResolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106005 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 246.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002845070
ELECTRON MICROSCOPYf_angle_d0.615661077
ELECTRON MICROSCOPYf_chiral_restr0.04516950
ELECTRON MICROSCOPYf_plane_restr0.00437818
ELECTRON MICROSCOPYf_dihedral_angle_d5.67825966

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