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- EMDB-29404: Acinetobacter baylyi LptB2FGC -

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Basic information

Entry
Database: EMDB / ID: EMD-29404
TitleAcinetobacter baylyi LptB2FGC
Map dataAcinetobacter baylyi LptB2FGC
Sample
  • Complex: Inner membrane lipopolysaccharide transporter composed of LptF, LptG, LptC and two molecules of LptB.
    • Protein or peptide: Lipopolysaccharide export system ATP-binding protein LptB
    • Protein or peptide: Lipopolysaccharide export system protein LptC
    • Protein or peptide: Lipopolysaccharide export system permease protein LptF
    • Protein or peptide: LPS export ABC transporter permease LptG
Keywordslipopolysaccharide / ABC / ATPase / antibiotic / macrocyclic peptide / gram-negative bacteria / ESKAPE / LIPID TRANSPORT
Function / homology
Function and homology information


ATP-binding cassette (ABC) transporter complex / transmembrane transport / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter ...Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / ABC transporter-like, conserved site / ABC transporters family signature. / 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
Lipopolysaccharide export system ATP-binding protein LptB / Permease / Lipopolysaccharide export system permease protein LptF
Similarity search - Component
Biological speciesAcinetobacter baylyi ADP1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPahil KS / Gilman MSA / Kruse AC / Kahne D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI149778 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI081059 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI153358 United States
CitationJournal: Nature / Year: 2024
Title: A new antibiotic traps lipopolysaccharide in its intermembrane transporter.
Authors: Karanbir S Pahil / Morgan S A Gilman / Vadim Baidin / Thomas Clairfeuille / Patrizio Mattei / Christoph Bieniossek / Fabian Dey / Dieter Muri / Remo Baettig / Michael Lobritz / Kenneth ...Authors: Karanbir S Pahil / Morgan S A Gilman / Vadim Baidin / Thomas Clairfeuille / Patrizio Mattei / Christoph Bieniossek / Fabian Dey / Dieter Muri / Remo Baettig / Michael Lobritz / Kenneth Bradley / Andrew C Kruse / Daniel Kahne /
Abstract: Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature ...Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
History
DepositionJan 7, 2023-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29404.png

Downloads & links

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Map

FileDownload / File: emd_29404.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAcinetobacter baylyi LptB2FGC
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.9785406 - 1.6107458
Average (Standard dev.)0.00009621132 (±0.04049107)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 267.3 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Acinetobacter baylyi LptB2FGC with the detergent micelle and...

Fileemd_29404_additional_1.map
AnnotationAcinetobacter baylyi LptB2FGC with the detergent micelle and periplasmic domains masked out.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_29404_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_29404_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Inner membrane lipopolysaccharide transporter composed of LptF, L...

EntireName: Inner membrane lipopolysaccharide transporter composed of LptF, LptG, LptC and two molecules of LptB.
Components
  • Complex: Inner membrane lipopolysaccharide transporter composed of LptF, LptG, LptC and two molecules of LptB.
    • Protein or peptide: Lipopolysaccharide export system ATP-binding protein LptB
    • Protein or peptide: Lipopolysaccharide export system protein LptC
    • Protein or peptide: Lipopolysaccharide export system permease protein LptF
    • Protein or peptide: LPS export ABC transporter permease LptG

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Supramolecule #1: Inner membrane lipopolysaccharide transporter composed of LptF, L...

SupramoleculeName: Inner membrane lipopolysaccharide transporter composed of LptF, LptG, LptC and two molecules of LptB.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Molecular weightTheoretical: 159.85 KDa

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Macromolecule #1: Lipopolysaccharide export system ATP-binding protein LptB

MacromoleculeName: Lipopolysaccharide export system ATP-binding protein LptB
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Molecular weightTheoretical: 27.934129 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEQIAQQQPQ TLCIKHLAKN YSKRWVVKDV SFEMQSGQIV GLLGPNGAGK TTSFYMVVGL VRMDKGEIHL DNLDLSDLAM HERARKGIG YLPQEASIFR KLTIAENIMA ILETRKDLNK QQRQQRLQEL LNDFKITHIK DSLGMSVSGG ERRRAEIARA L AADPKFML ...String:
MEQIAQQQPQ TLCIKHLAKN YSKRWVVKDV SFEMQSGQIV GLLGPNGAGK TTSFYMVVGL VRMDKGEIHL DNLDLSDLAM HERARKGIG YLPQEASIFR KLTIAENIMA ILETRKDLNK QQRQQRLQEL LNDFKITHIK DSLGMSVSGG ERRRAEIARA L AADPKFML LDEPFAGVDP ISVGDIKDII RNLKDRGIGV LITDHNVRET LAICEHAYIV SEGAVIAEGS PQDILENEQV RK VYLGDDF TV

UniProtKB: Lipopolysaccharide export system ATP-binding protein LptB

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Macromolecule #2: Lipopolysaccharide export system protein LptC

MacromoleculeName: Lipopolysaccharide export system protein LptC / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Molecular weightTheoretical: 1.805216 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)

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Macromolecule #3: Lipopolysaccharide export system permease protein LptF

MacromoleculeName: Lipopolysaccharide export system permease protein LptF
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Molecular weightTheoretical: 41.564273 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIIRRYLVKQ VVSTSLVVIA LLTLIMMGGR LIKYFGVAAQ GRLDAGVLFS IIGYRMPEFL TLILPLGFFI GLMLVFGRLY VDHEMAVLN GSGISRIRLG QLLIPLALVF LVIQGILMLW MTPWGLRQFD QLSSSQAVRT GFDLVRPKEF ISSGPYTIYA G DLSEDRKN ...String:
MIIRRYLVKQ VVSTSLVVIA LLTLIMMGGR LIKYFGVAAQ GRLDAGVLFS IIGYRMPEFL TLILPLGFFI GLMLVFGRLY VDHEMAVLN GSGISRIRLG QLLIPLALVF LVIQGILMLW MTPWGLRQFD QLSSSQAVRT GFDLVRPKEF ISSGPYTIYA G DLSEDRKN LKDIFFYQRA QKEGKPDVMI LAKEATRVVM ENETANVVDL IQGRRYEIYP GKAKYSQAEF QRYRLRLEND KS ATFETDK VEALPSSKLW NKWNDPVIAS EMGWRVFGPF TIVIALMMAV ALCEVSPRQG RYYRLIPAIF IFASLIVLLI AIR TRISRD ELGVWAYPAA LAVYGIAAAL FSRKQKLAPK IKKQIKRVRA

UniProtKB: Lipopolysaccharide export system permease protein LptF

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Macromolecule #4: LPS export ABC transporter permease LptG

MacromoleculeName: LPS export ABC transporter permease LptG / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Molecular weightTheoretical: 40.080621 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLARRIVAKH VTKTTALAML GTTIVLVILQ VLFTYLGELS NLKADYSAWQ AFLYVLWGAP RYLYEILPIS ALIGAILGLG TLASNSELI VMRSVGISLW RIVGWVIRSA LVLVLLSFAL SEWVVPYTNE RANSVKSHQS VAALGEVRGY WSREGQRFIY V DYANSQGQ ...String:
MLARRIVAKH VTKTTALAML GTTIVLVILQ VLFTYLGELS NLKADYSAWQ AFLYVLWGAP RYLYEILPIS ALIGAILGLG TLASNSELI VMRSVGISLW RIVGWVIRSA LVLVLLSFAL SEWVVPYTNE RANSVKSHQS VAALGEVRGY WSREGQRFIY V DYANSQGQ LKRIQVVDFD DNYRLKSVTN AEQGQFVKDG QWLLNHSQQM AIQGQGDAVL ANAAKQPFSL ALQPKYVHMV TI DPEDLSF SQLVSFMNYM REYSQVPKTY QLAFWKKVAS PFALITLVLV ACSFIFGPLR QQSMGFRLVI ALFIGLGFYY LQD FLGYAS LVYNPSPAWF VLGPIVLMFV AGSYLLYRAR

UniProtKB: Permease

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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

Concentration7 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
300.0 mMNaClSodium chlorideSodium chloride
20.0 mMNH2C(CH2OH)3Tris pH 7.4
0.02 %C56H92O25glyco-diosgenin
0.25 mMC9H21O3Ptris(hydroxypropyl)phosphine
8.0 mMC3H4N2imidazole

Details: 300 mM NaCl, 20 mM Tris [pH 7.4], 0.02% GDN, 0.25 mM tris(hydroxypropyl)phosphine, 8 mM imidazole
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse as determined by SEC.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6000000
Startup modelType of model: OTHER
Details: Ab initio model generated based on the data using cryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 65285
FSC plot (resolution estimation)

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