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- EMDB-43965: Cryo-EM reconstruction of a Staphylococcus aureus oleate hydratas... -

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Basic information

Entry
Database: EMDB / ID: EMD-43965
TitleCryo-EM reconstruction of a Staphylococcus aureus oleate hydratase (OhyA) assembly of dimers bound to a liposome
Map data
Sample
  • Complex: Assembly of dimers of OhyA bound to a liposome
    • Protein or peptide: Oleate hydratase
Keywordsoleate hydratase / phospholipids / peripheral membrane protein / OhyA / 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: 3.1 Å
AuthorsOldham ML / Qayyum MZ / Radka CD
Funding support United States, 1 items
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
DepositionMar 6, 2024-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43965.png

Downloads & links

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Map

FileDownload / File: emd_43965.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 640 pix.
= 668.16 Å		1.04 Å/pix.
x 640 pix.
= 668.16 Å		1.04 Å/pix.
x 640 pix.
= 668.16 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.044 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.225
Minimum - Maximum-0.41739178 - 1.1111897
Average (Standard dev.)0.0004504447 (±0.0237612)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 668.16003 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_43965_additional_1.map
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Half map: #1

Fileemd_43965_half_map_1.map
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Half map: #2

Fileemd_43965_half_map_2.map
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Sample components

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Entire : Assembly of dimers of OhyA bound to a liposome

EntireName: Assembly of dimers of OhyA bound to a liposome
Components
  • Complex: Assembly of dimers of OhyA bound to a liposome
    • Protein or peptide: Oleate hydratase

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Supramolecule #1: Assembly of dimers of OhyA bound to a liposome

SupramoleculeName: Assembly of dimers of OhyA bound to a liposome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 977.4 KDa

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Macromolecule #1: Oleate hydratase

MacromoleculeName: Oleate hydratase / type: protein_or_peptide / ID: 1 / Number of copies: 14 / 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:
ConcentrationNameFormula
25.0 mMHEPES
150.0 mMSodium ChlorideNaCl
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details3mg/mL OhyA added to 15mM prepared POPC:POPG liposomes

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMax: 100.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 15784 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 79000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8ur3

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 87806
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8ur3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9axe:
Cryo-EM reconstruction of a Staphylococcus aureus oleate hydratase (OhyA) assembly of dimers bound to a liposome

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