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- EMDB-42480: Cryo-EM reconstruction of Staphylococcus aureus Oleate hydratase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-42480
TitleCryo-EM reconstruction of Staphylococcus aureus Oleate hydratase (OhyA) dimer with an ordered C-terminal membrane-association domain
Map data
Sample
  • Complex: Homodimer of OhyA
    • Protein or peptide: Oleate hydratase
Keywordsoleate hydratase (OhyA) / phospholipids / membrane binding domain / amphipathic helices / interfacial enzyme / peripheral membrane protein / HYDROLASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsOldham ML / Qayyum MZ
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42480.png

Downloads & links

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Map

FileDownload / File: emd_42480.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.044 Å
Density
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.24177748 - 0.9401327
Average (Standard dev.)-0.00021506216 (±0.021121275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 271.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer map

Fileemd_42480_additional_1.map
AnnotationDeepEMhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42480_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42480_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimer of OhyA

EntireName: Homodimer of OhyA
Components
  • Complex: Homodimer of OhyA
    • Protein or peptide: Oleate hydratase

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Supramolecule #1: Homodimer of OhyA

SupramoleculeName: Homodimer of OhyA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 139.343 KDa

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Macromolecule #1: Oleate hydratase

MacromoleculeName: Oleate hydratase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 69.892719 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MYYSYGNYEA FARPKKPENV ENKSAYLIGS GLASLAAACF LIRDGQMEGS KIHILEELPK AGGSLDGEN MPLKGYVVRG GREMENHFEC LWDLFRSIPS LEIDNASVLD EFYWLNKEDP NYSRCRVIEK QGQRLVTDGD F TLTKTAIK ...String:
MGSSHHHHHH SSGLVPRGSH MYYSYGNYEA FARPKKPENV ENKSAYLIGS GLASLAAACF LIRDGQMEGS KIHILEELPK AGGSLDGEN MPLKGYVVRG GREMENHFEC LWDLFRSIPS LEIDNASVLD EFYWLNKEDP NYSRCRVIEK QGQRLVTDGD F TLTKTAIK EILDLCLTNE EDLDDVKITD VFSDDFFNSN FWIYWKTMFA FEPWHSAMEM RRYLMRFVHH ISGLADFSAL KF TKYNQYE SLVLPMVEYL KSHGVQFEYD VKVEDIKIDV TTSQKIAREI LIDRNGNAES IKLTINDLVF VTNGSITESS TYG DNDTPA PPTDELGGSW TLWKNLARQS PEFGNPDKFC QNIPKKSWFV SATSTTNNKE IIDTIESICK RDPLAGKTVT GGII TINDS AWQMSFTINR QQQFKDQPEN EISTWIYALY SDVNGDYIKK PITECSGNEI CQEWLYHLGV STDKIEDLAK HASNT IPVY MPYITSYFMT RAIGDRPLVV PHQSQNLAFI GNFAETERDT VFTTEYSVRT AMEAVYQLLN IDRGIPEVIN SPFDLR VLM DAIYELNDHQ DLREITKDSK MQKLALAGFL KKIKGTYIES LLKEHKLL

UniProtKB: Myosin-cross-reactive antigen

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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

Concentration3 mg/mL
BufferpH: 7.3
Component:
ConcentrationName
25.0 mMHEPES
150.0 mMSodium Chloride
50.0 uMLauryl Maltose Neopentyl Glycol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 79000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMax: 100.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3248 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3065496
Details: template picking using templates generated from model map reconstructed from pdb 7kav
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7kav

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. 4) / Details: 3D classification to remove Dimer of Dimers
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 572989
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7kav


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: initial model was a crystal structure
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8ur3:
Cryo-EM reconstruction of Staphylococcus aureus Oleate hydratase (OhyA) dimer with an ordered C-terminal membrane-association domain

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