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- EMDB-35203: Cryo-EM structure of Pseudomonas aeruginosa FtsE(WT)X complex in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-35203
TitleCryo-EM structure of Pseudomonas aeruginosa FtsE(WT)X complex in peptidisc
Map data
Sample
  • Complex: FtsEX
    • Protein or peptide: Cell division ATP-binding protein FtsE
    • Protein or peptide: Cell division protein FtsX
KeywordsType VII ABC transporter / divisome / PG hydrolysis / MEMBRANE PROTEIN
Function / homology
Function and homology information


cell cycle / cell division / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
: / Cell division protein FtsE, ATP-binding / Cell division protein FtsX / FtsX, extracellular domain / FtsX extracellular domain / ABC transporter, lipoprotein release, LolD / ABC3 transporter permease protein domain / FtsX-like permease family / ABC transporter / ABC transporter-like, ATP-binding domain ...: / Cell division protein FtsE, ATP-binding / Cell division protein FtsX / FtsX, extracellular domain / FtsX extracellular domain / ABC transporter, lipoprotein release, LolD / ABC3 transporter permease protein domain / FtsX-like permease family / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cell division ATP-binding protein FtsE / Cell division protein FtsX
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsXin X / Jianwei L / Min L
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)A-0008412-00-00 Singapore
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Mechanistic insights into the regulation of cell wall hydrolysis by FtsEX and EnvC at the bacterial division site.
Authors: Xin Xu / Jianwei Li / Wan-Zhen Chua / Martin A Pages / Jian Shi / Juan A Hermoso / Thomas Bernhardt / Lok-To Sham / Min Luo /
Abstract: The peptidoglycan (PG) cell wall produced by the bacterial division machinery is initially shared between the daughters and must be split to promote cell separation and complete division. In gram- ...The peptidoglycan (PG) cell wall produced by the bacterial division machinery is initially shared between the daughters and must be split to promote cell separation and complete division. In gram-negative bacteria, enzymes that cleave PG called amidases play major roles in the separation process. To prevent spurious cell wall cleavage that can lead to cell lysis, amidases like AmiB are autoinhibited by a regulatory helix. Autoinhibition is relieved at the division site by the activator EnvC, which is in turn regulated by the ATP-binding cassette (ABC) transporter-like complex called FtsEX. EnvC is also known to be autoinhibited by a regulatory helix (RH), but how its activity is modulated by FtsEX and the mechanism by which it activates the amidases have remained unclear. Here, we investigated this regulation by determining the structure of FtsEX alone with or without bound ATP, in complex with EnvC, and in a FtsEX-EnvC-AmiB supercomplex. In combination with biochemical studies, the structures reveal that ATP binding is likely to activate FtsEX-EnvC and promote its association with AmiB. Furthermore, the AmiB activation mechanism is shown to involve a RH rearrangement. In the activated state of the complex, the inhibitory helix of EnvC is released, freeing it to associate with the RH of AmiB, which liberates its active site for PG cleavage. These regulatory helices are found in many EnvC proteins and amidases throughout gram-negative bacteria, suggesting that the activation mechanism is broadly conserved and a potential target for lysis-inducing antibiotics that misregulate the complex.
History
DepositionJan 29, 2023-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJun 7, 2023-
Current statusJun 7, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35203.png

Downloads & links

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Map

FileDownload / File: emd_35203.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.858 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.13769412 - 0.75608224
Average (Standard dev.)-0.00074842543 (±0.029088832)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 274.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35203_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35203_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FtsEX

EntireName: FtsEX
Components
  • Complex: FtsEX
    • Protein or peptide: Cell division ATP-binding protein FtsE
    • Protein or peptide: Cell division protein FtsX

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Supramolecule #1: FtsEX

SupramoleculeName: FtsEX / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 124 kDa/nm

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Macromolecule #1: Cell division protein FtsX

MacromoleculeName: Cell division protein FtsX / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 36.964359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSANDLPRGP EEGAPERKTR EKPSQEQTDW SGSFSAYLES HRASLVDSLR RLFGHPFGSF FTCLVMGITL SLPMGLSLLL NNVERLGGS WQRAAQISLF LDLKTSENQG QDLREQIERL PDVIEAQLIS REQALSELQE QSGLGEALKE LPENPLPPVI S VTPKQIDR ...String:
MSANDLPRGP EEGAPERKTR EKPSQEQTDW SGSFSAYLES HRASLVDSLR RLFGHPFGSF FTCLVMGITL SLPMGLSLLL NNVERLGGS WQRAAQISLF LDLKTSENQG QDLREQIERL PDVIEAQLIS REQALSELQE QSGLGEALKE LPENPLPPVI S VTPKQIDR AGLEALRQQL AELPHVQQAQ LDLVWVERLS AILKLGERFV FGLTILLVLT LLLVVGNTIR LHIENRRNEI EV IKLVGGT DGYVRRPFLY MGALYGLGAG ILSWALLAYS LNWLNGSVVN LSGLYGSDFG LQGVPLDDGL SLTVGAVLLG WVG AWLAVA RHLRELAPR

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Macromolecule #2: Cell division ATP-binding protein FtsE

MacromoleculeName: Cell division ATP-binding protein FtsE / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 24.649666 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIRFEQVGKR YPNGHVGLHE VSFRVHRGEI LFVTGHSGAG KSTLLRLILA MERPTSGKLL LGGQDLGRIT TAQIPFLRRQ IGVVFQNHQ LLTDRTVADN IALPLQILGM PKPEIAKRVA SALERVNLKE KGEALPSDLS TGQQQRVGIA RAIVHQPALL L ADEPTGNL ...String:
MIRFEQVGKR YPNGHVGLHE VSFRVHRGEI LFVTGHSGAG KSTLLRLILA MERPTSGKLL LGGQDLGRIT TAQIPFLRRQ IGVVFQNHQ LLTDRTVADN IALPLQILGM PKPEIAKRVA SALERVNLKE KGEALPSDLS TGQQQRVGIA RAIVHQPALL L ADEPTGNL DPRLASEIMG VFEDINRLGT TVLIASHDLA LIARMRHRML TLQRGRIIAD REDEA

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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: 7.5 / Details: 25 mM Tris, pH 7.5, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 28977
FSC plot (resolution estimation)

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