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- PDB-8ur8: Cryo-EM reconstruction of Staphylococcus aureus oleate hydratase ... -

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Basic information

Entry
Database: PDB / ID: 8ur8
TitleCryo-EM reconstruction of Staphylococcus aureus oleate hydratase (OhyA) dimer of dimers
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: 2.97 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)AI166116-03 United States
CitationJournal: J Struct Biol / Year: 2024
Title: Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer.
Authors: Michael L Oldham / M Zuhaib Qayyum / Ravi C Kalathur / Charles O Rock / Christopher D Radka /
Abstract: Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen ...Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.
History
DepositionOct 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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 / _em_admin.last_update
Revision 1.2Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update
Revision 1.3Sep 11, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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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
C: Oleate hydratase
D: Oleate hydratase


Theoretical massNumber of molelcules
Total (without water)279,5714
Polymers279,5714
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.: 4
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: Dimer of Dimers of OhyA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.278686 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)Star
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2150 mMSodium ChlorideNaCl1
31 mMLauryl 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: 276

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPUimage acquisition
4cryoSPARC4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
13cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 372852
Details: template picking using templates generated from model map reconstructed from pdb 7kav
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73589 / 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_d0.00619184
ELECTRON MICROSCOPYf_angle_d0.625996
ELECTRON MICROSCOPYf_dihedral_angle_d4.7792504
ELECTRON MICROSCOPYf_chiral_restr0.0492852
ELECTRON MICROSCOPYf_plane_restr0.0043336

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