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- EMDB-35213: Cryo-EM structure of Pseudomonas aeruginosa FtsE(E163Q)X/EnvC/Ami... -

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Basic information

Entry
Database: EMDB / ID: EMD-35213
TitleCryo-EM structure of Pseudomonas aeruginosa FtsE(E163Q)X/EnvC/AmiB complex with ATP in peptidisc
Map data
Sample
  • Complex: FtsEE163QX/EnvC/AmiB-ATP
    • Protein or peptide: FstEE163Q
    • Protein or peptide: FtsX
    • Protein or peptide: EnvC
    • Protein or peptide: AmiB
Keywordstype VII ABC transporter / divisome / PG hydrolysis / MEMBRANE PROTEIN
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.7 Å
AuthorsXu X / Li J / Luo M
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 31, 2023-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35213.png

Downloads & links

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Map

FileDownload / File: emd_35213.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.716 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.44721124 - 2.8198323
Average (Standard dev.)-0.002181133 (±0.05272518)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 658.944 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35213_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_35213_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : FtsEE163QX/EnvC/AmiB-ATP

EntireName: FtsEE163QX/EnvC/AmiB-ATP
Components
  • Complex: FtsEE163QX/EnvC/AmiB-ATP
    • Protein or peptide: FstEE163Q
    • Protein or peptide: FtsX
    • Protein or peptide: EnvC
    • Protein or peptide: AmiB

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Supramolecule #1: FtsEE163QX/EnvC/AmiB-ATP

SupramoleculeName: FtsEE163QX/EnvC/AmiB-ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 216 KDa

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Macromolecule #1: FstEE163Q

MacromoleculeName: FstEE163Q / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIRFEQVGKR YPNGHVGLHE VSFRVHRGEI LFVTGHSGAG KSTLLRLILA MERPTSGKLL LGGQDLGRIT TAQIPFLRRQ IGVVFQNHQL LTDRTVADNI ALPLQILGMP KPEIAKRVAS ALERVNLKEK GEALPSDLST GQQQRVGIAR AIVHQPALLL ADQPTGNLDP ...String:
MIRFEQVGKR YPNGHVGLHE VSFRVHRGEI LFVTGHSGAG KSTLLRLILA MERPTSGKLL LGGQDLGRIT TAQIPFLRRQ IGVVFQNHQL LTDRTVADNI ALPLQILGMP KPEIAKRVAS ALERVNLKEK GEALPSDLST GQQQRVGIAR AIVHQPALLL ADQPTGNLDP RLASEIMGVF EDINRLGTTV LIASHDLALI ARMRHRMLTL QRGRIIADRE DEA

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Macromolecule #2: FtsX

MacromoleculeName: FtsX / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSANDLPRGP EEGAPERKTR EKPSQEQTDW SGSFSAYLES HRASLVDSLR RLFGHPFGSF FTCLVMGITL SLPMGLSLLL NNVERLGGSW QRAAQISLFL DLKTSENQGQ DLREQIERLP DVIEAQLISR EQALSELQEQ SGLGEALKEL PENPLPPVIS VTPKQIDRAG ...String:
MSANDLPRGP EEGAPERKTR EKPSQEQTDW SGSFSAYLES HRASLVDSLR RLFGHPFGSF FTCLVMGITL SLPMGLSLLL NNVERLGGSW QRAAQISLFL DLKTSENQGQ DLREQIERLP DVIEAQLISR EQALSELQEQ SGLGEALKEL PENPLPPVIS VTPKQIDRAG LEALRQQLAE LPHVQQAQLD LVWVERLSAI LKLGERFVFG LTILLVLTLL LVVGNTIRLH IENRRNEIEV IKLVGGTDGY VRRPFLYMGA LYGLGAGILS WALLAYSLNW LNGSVVNLSG LYGSDFGLQG VPLDDGLSLT VGAVLLGWVG AWLAVARHLR ELAPR

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Macromolecule #3: EnvC

MacromoleculeName: EnvC / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DERADTQRQL EQTQKDIGEL KKLLDGIQQE KSGVQKQLKS TETEMGDLEK QIKALQDELD KSEAELKRLD GEKKKLQDAR IEQQRLLAIQ ARAAYQSGRE EYLKLLLNQE HPEKFSRTLT YYDYINKARL EQLASFNETL RQLANVEQDI SAQKAEQLSK QGELDSRREA ...String:
DERADTQRQL EQTQKDIGEL KKLLDGIQQE KSGVQKQLKS TETEMGDLEK QIKALQDELD KSEAELKRLD GEKKKLQDAR IEQQRLLAIQ ARAAYQSGRE EYLKLLLNQE HPEKFSRTLT YYDYINKARL EQLASFNETL RQLANVEQDI SAQKAEQLSK QGELDSRREA LAATRKERQQ ALAKLNSDYR ERDQKLKSRQ QDQAELAKVL RTIEETLARQ AREAAAAAER ERQRALAAER ERARQQQAAP GRVTSPPREP APGPLVSSTG AVYGGAFGSA RGKLPWPVNG RVVARFGSQR GDDPRAKWDG VLISASAGST VRAVHGGRVV FADWLRGAGL LVILDHGGGY LSLYGHNQSL LKDAGDTVKA GDPIATVGTS GGQSSPAVYF AIRHQGRPAD PTTWCRAQG

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Macromolecule #4: AmiB

MacromoleculeName: AmiB / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QIKSVRIWRA PDNTRLVFDL SGPVQHSLFT LAAPNRIVID VSGAQLATQL NGLKLGNTPI TAVRSAQRTP NDLRMVLDLS AQVTPKSFVL PPNQQYGNRL VVDLYDQGAD LTPDVPATPT PSVPVTPVTP TQPVAKLPLP TKGGTRDIVI AIDAGHGGED PGALGPGGLH ...String:
QIKSVRIWRA PDNTRLVFDL SGPVQHSLFT LAAPNRIVID VSGAQLATQL NGLKLGNTPI TAVRSAQRTP NDLRMVLDLS AQVTPKSFVL PPNQQYGNRL VVDLYDQGAD LTPDVPATPT PSVPVTPVTP TQPVAKLPLP TKGGTRDIVI AIDAGHGGED PGALGPGGLH EKNITLSIAR ELQRQINQVR GYRAELTRTG DYFIPLRKRT EIARKKGADL FVSIHADAAP SRSAFGASVF ALSDRGATSE TARWLADSEN RSDLIGGDGS VSLGDKDQML AGVLLDLSMT ATLSSSLDVG HKVLTNVGRI TSLHKRRVEQ AGFMVLKSPD IPSILVETGF ISNVNESRKL ASASHQQALA RSITSGIRQY FQQSPPPGTY IASLRAQGKL SMGPREHVVR PGETLAMIAQ RYEVSMAALR SSNSLSSDNL KVGQALSIPS TALAAQ

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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, 2mM ATP/MgCl2
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: 19.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 37628
FSC plot (resolution estimation)

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