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- EMDB-17217: Structure of Staphylococcus aureus FloA protein -

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Basic information

Entry
Database: EMDB / ID: EMD-17217
TitleStructure of Staphylococcus aureus FloA protein
Map dataS.aureus FloA cryo-EM map
Sample
  • Complex: FloA protein monomer
    • Protein or peptide: Flotillin-like protein FloA
Keywordssmall membrane protein / chapperone / lipid rafts / antibiotic resistance / staphylococcus / membrane microdomains / MEMBRANE PROTEIN
Function / homologyFlotillin-like protein FloA / Flotillin-like protein FloA / membrane raft / plasma membrane / Flotillin-like protein FloA
Function and homology information
Biological speciesStaphylococcus aureus subsp. aureus N315 (bacteria) / Staphylococcus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsUkleja M / Kricks L / Torrens G / Peschiera I / Rodrigues-Lopes I / Krupka M / Garcia Fernandez J / del Campo R / Eulalio A / Mateus A ...Ukleja M / Kricks L / Torrens G / Peschiera I / Rodrigues-Lopes I / Krupka M / Garcia Fernandez J / del Campo R / Eulalio A / Mateus A / Lopez Bravo M / Rico AI / Cava F / Lopez D
Funding support Sweden, Spain, 2 items
OrganizationGrant numberCountry
Swedish Research Council Sweden
Spanish Ministry of Science, Innovation, and Universities Spain
CitationJournal: Nat Commun / Year: 2024
Title: Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.
Authors: Marta Ukleja / Lara Kricks / Gabriel Torrens / Ilaria Peschiera / Ines Rodrigues-Lopes / Marcin Krupka / Julia García-Fernández / Roberto Melero / Rosa Del Campo / Ana Eulalio / André ...Authors: Marta Ukleja / Lara Kricks / Gabriel Torrens / Ilaria Peschiera / Ines Rodrigues-Lopes / Marcin Krupka / Julia García-Fernández / Roberto Melero / Rosa Del Campo / Ana Eulalio / André Mateus / María López-Bravo / Ana I Rico / Felipe Cava / Daniel Lopez /
Abstract: The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the ...The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.
History
DepositionApr 26, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17217.png

Downloads & links

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Map

FileDownload / File: emd_17217.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationS.aureus FloA cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.58 Å/pix.
x 320 pix.
= 185.6 Å		0.58 Å/pix.
x 320 pix.
= 185.6 Å		0.58 Å/pix.
x 320 pix.
= 185.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.58 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0237
Minimum - Maximum-0.013969864 - 0.24424292
Average (Standard dev.)0.0011963352 (±0.0053198254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 185.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_17217_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_17217_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FloA protein monomer

EntireName: FloA protein monomer
Components
  • Complex: FloA protein monomer
    • Protein or peptide: Flotillin-like protein FloA

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Supramolecule #1: FloA protein monomer

SupramoleculeName: FloA protein monomer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: S.aureus FloA protein, full lenght
Source (natural)Organism: Staphylococcus aureus subsp. aureus N315 (bacteria)
Molecular weightTheoretical: 35 KDa

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Macromolecule #1: Flotillin-like protein FloA

MacromoleculeName: Flotillin-like protein FloA / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Staphylococcus (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MFSLSFIVIA VIIIVALLIL FSFVPIGLWI SALAAGVHVG IGTLVGMRLR RVSPRKVIAP LIKAHKAGLA LTTNQLESHY LAGGNVDRVV DANIAAQRAD IDLPFERAAA IDLAGRDVLE AVQMSVNPKV IETPFIAGVA MNGIEVKAKA RITVRANIAR LVGGAGEETI ...String:
MFSLSFIVIA VIIIVALLIL FSFVPIGLWI SALAAGVHVG IGTLVGMRLR RVSPRKVIAP LIKAHKAGLA LTTNQLESHY LAGGNVDRVV DANIAAQRAD IDLPFERAAA IDLAGRDVLE AVQMSVNPKV IETPFIAGVA MNGIEVKAKA RITVRANIAR LVGGAGEETI IARVGEGIVS TIGSSKHHTE VLENPDNISK TVLSKGLDSG TAFEILSIDI ADVDISKNIG ADLQTEQALA DKNIAQAKAE ERRAMAVATE QEMKARVQEM HAKVVEAESE VPLAMAEALR SGNISVKDYY NLKNIEADTG MRNAINKRTD QSDDESPEH

UniProtKB: Flotillin-like protein FloA

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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

Concentration0.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMTrisTris
300.0 mMNaClNaCl
0.05 %DDMDDM
GridModel: UltrAuFoil R2/2 / Support film - Material: GOLD / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsFull lenght FloA

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 9653 / Average exposure time: 2.33 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 215000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 478555
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.0) / Software - details: Scipion / Number images used: 118042
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC / Software - details: Scipion / Details: ab initio reconstruction protocol from cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.0.0) / Software - details: Scipion / Details: Local refinement in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation criteria

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