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- EMDB-32064: The oligomerized complex of hemolysin AF3 mutant protomers. -

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Entry
Database: EMDB / ID: EMD-32064
TitleThe oligomerized complex of hemolysin AF3 mutant protomers.
Map data
Sample
  • Complex: The oligomerized complex of hemolysin AF3 mutant protomers.
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsGhanem N / Hashimoto T / Yokoyama T / Tanaka Y
Funding support Japan, 7 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H05492 Japan
Japan Society for the Promotion of Science (JSPS)19KK0178 Japan
Japan Society for the Promotion of Science (JSPS)19H03511 Japan
Japan Society for the Promotion of Science (JSPS)19H03040 Japan
Japan Society for the Promotion of Science (JSPS)19H02831 Japan
Japan Society for the Promotion of Science (JSPS)19H02519 Japan
Japan Society for the Promotion of Science (JSPS)19H00918 Japan
CitationJournal: FEBS J / Year: 2022
Title: Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain.
Authors: Nouran Ghanem / Natsuki Kanagami / Takashi Matsui / Kein Takeda / Jun Kaneko / Yasuyuki Shiraishi / Christian A Choe / Tomomi Uchikubo-Kamo / Mikako Shirouzu / Tsubasa Hashimoto / Tomohisa ...Authors: Nouran Ghanem / Natsuki Kanagami / Takashi Matsui / Kein Takeda / Jun Kaneko / Yasuyuki Shiraishi / Christian A Choe / Tomomi Uchikubo-Kamo / Mikako Shirouzu / Tsubasa Hashimoto / Tomohisa Ogawa / Tomoaki Matsuura / Po-Ssu Huang / Takeshi Yokoyama / Yoshikazu Tanaka /
Abstract: Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble ...Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane β-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a β-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the β-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT β-barrels for use in further promising applications.
History
DepositionOct 17, 2021-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateOct 12, 2022-
Current statusOct 12, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_32064.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.034023006 - 0.069316916
Average (Standard dev.)0.0018872132 (±0.011680575)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : The oligomerized complex of hemolysin AF3 mutant protomers.

EntireName: The oligomerized complex of hemolysin AF3 mutant protomers.
Components
  • Complex: The oligomerized complex of hemolysin AF3 mutant protomers.

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Supramolecule #1: The oligomerized complex of hemolysin AF3 mutant protomers.

SupramoleculeName: The oligomerized complex of hemolysin AF3 mutant protomers.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Staphylococcus aureus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl acetate

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 40.0 e/Å2

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 6002

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