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- PDB-8ur3: Cryo-EM reconstruction of Staphylococcus aureus Oleate hydratase ... -

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Basic information

Entry
Database: PDB / ID: 8ur3
TitleCryo-EM reconstruction of Staphylococcus aureus Oleate hydratase (OhyA) dimer with an ordered C-terminal membrane-association domain
ComponentsOleate hydratase
KeywordsHYDROLASE / oleate hydratase (OhyA) / phospholipids / membrane binding domain / amphipathic helices / interfacial enzyme / peripheral membrane protein
Function / homologyoleate hydratase / oleate hydratase activity / Oleate hydratase / MCRA family / FAD binding / fatty acid metabolic process / FAD/NAD(P)-binding domain superfamily / Myosin-cross-reactive antigen
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsOldham, M.L. / Qayyum, M.Z.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)4R00AI166116-03 United States
CitationJournal: J Biol Chem / Year: 2024
Title: The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.
Authors: Christopher D Radka / Christy R Grace / Hale S Hasdemir / Yupeng Li / Carlos C Rodriguez / Patrick Rodrigues / Michael L Oldham / M Zuhaib Qayyum / Aaron Pitre / William J MacCain / Ravi C ...Authors: Christopher D Radka / Christy R Grace / Hale S Hasdemir / Yupeng Li / Carlos C Rodriguez / Patrick Rodrigues / Michael L Oldham / M Zuhaib Qayyum / Aaron Pitre / William J MacCain / Ravi C Kalathur / Emad Tajkhorshid / Charles O Rock /
Abstract: The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the ...The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.
History
DepositionOct 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 14, 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: Oleate hydratase
B: Oleate hydratase


Theoretical massNumber of molelcules
Total (without water)139,7852
Polymers139,7852
Non-polymers00
Water00
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 Oleate hydratase / Myosin-cross-reactive antigen


Mass: 69892.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: DD547_00094, DQV53_00770, EP54_06595, EQ90_12415, G0V24_00735, G0X12_00605, G0Z18_00840, G6Y10_10285, GO746_00220, GO803_11440, GO805_08895, GO821_10135, GO894_07145, GO942_14045, HMPREF3211_ ...Gene: DD547_00094, DQV53_00770, EP54_06595, EQ90_12415, G0V24_00735, G0X12_00605, G0Z18_00840, G6Y10_10285, GO746_00220, GO803_11440, GO805_08895, GO821_10135, GO894_07145, GO942_14045, HMPREF3211_02399, NCTC10654_00136, RK64_00980
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star / References: UniProt: A0A0D6GJV1

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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: Homodimer of OhyA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.139343 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)Star
Buffer solutionpH: 7.3
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMSodium Chloride1
350 uMLauryl Maltose Neopentyl Glycol1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 FIELD / Nominal magnification: 79000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3248

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPUimage acquisition
4cryoSPARC4CTF correction
7Cootmodel fitting
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3065496
Details: template picking using templates generated from model map reconstructed from pdb 7kav
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 572989 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7kav

7kav


Accession code: 7kav / Details: initial model was a crystal structure / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039592
ELECTRON MICROSCOPYf_angle_d0.51112998
ELECTRON MICROSCOPYf_dihedral_angle_d4.211252
ELECTRON MICROSCOPYf_chiral_restr0.0451426
ELECTRON MICROSCOPYf_plane_restr0.0031668

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