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- EMDB-9125: Nucleotide-free Cryo-EM Structure of E.coli LptB2FGC -

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Basic information

Entry
Database: EMDB / ID: EMD-9125
TitleNucleotide-free Cryo-EM Structure of E.coli LptB2FGC
Map dataCryo-EM map of nucleotide-free LptB2FGC filtered to 4.2A with -200 b-factor
Sample
  • Complex: LptB2FGC
    • Protein or peptide: Lipopolysaccharide export system permease protein LptF
    • Protein or peptide: Lipopolysaccharide export system permease protein LptG
    • Protein or peptide: Lipopolysaccharide export system ATP-binding protein LptB
    • Protein or peptide: Lipopolysaccharide export system protein LptC
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
KeywordsABC transporter / lipopolysaccharide / LPS / nanodisc / MEMBRANE PROTEIN / HYDROLASE-TRANSPORT PROTEIN complex
Function / homology
Function and homology information


lipopolysaccharide transmembrane transporter activity / Translocases; Catalysing the translocation of carbohydrates and their derivatives; Linked to the hydrolysis of a nucleoside triphosphate / transporter complex / glycolipid transfer activity / lipopolysaccharide transport / Gram-negative-bacterium-type cell outer membrane assembly / ATP-binding cassette (ABC) transporter complex / transmembrane transport / outer membrane-bounded periplasmic space / ATP hydrolysis activity ...lipopolysaccharide transmembrane transporter activity / Translocases; Catalysing the translocation of carbohydrates and their derivatives; Linked to the hydrolysis of a nucleoside triphosphate / transporter complex / glycolipid transfer activity / lipopolysaccharide transport / Gram-negative-bacterium-type cell outer membrane assembly / ATP-binding cassette (ABC) transporter complex / transmembrane transport / outer membrane-bounded periplasmic space / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Lipopolysaccharide assembly, LptC-related / Lipopolysaccharide export system protein LptC / : / Lipopolysaccharide-assembly, LptC-related / 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 ...Lipopolysaccharide assembly, LptC-related / Lipopolysaccharide export system protein LptC / : / Lipopolysaccharide-assembly, LptC-related / 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 / Lipopolysaccharide export system permease protein LptG / Lipopolysaccharide export system protein LptC / Lipopolysaccharide export system permease protein LptF
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsOrlando BJ / Li Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122797 United States
CitationJournal: Nature / Year: 2019
Title: Structural basis of lipopolysaccharide extraction by the LptBFGC complex.
Authors: Yanyan Li / Benjamin J Orlando / Maofu Liao /
Abstract: In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide ...In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide from the inner to the outer membrane. The ATP-binding cassette transporter LptBFG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane. The mechanism of the LptBFG-LptC complex (LptBFGC) and the role of LptC in lipopolysaccharide transport are poorly understood. Here we characterize the structures of LptBFG and LptBFGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptBFGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptBFG, which represents a previously unknown regulatory mechanism for ATP-binding cassette transporters. Our results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptBFG in the inner membrane and Lpt protein interactions in the periplasm.
History
DepositionSep 19, 2018-
Header (metadata) releaseOct 17, 2018-
Map releaseApr 3, 2019-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.085
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.085
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mi7
  • Surface level: 0.085
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9125.png

Downloads & links

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Map

FileDownload / File: emd_9125.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of nucleotide-free LptB2FGC filtered to 4.2A with -200 b-factor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.085 / Movie #1: 0.085
Minimum - Maximum-0.2449101 - 0.37801304
Average (Standard dev.)-0.000043752403 (±0.015729474)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 236.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.160236.160236.160
α/β/γ90.00090.00090.000
start NX/NY/NZ-383-383-383
NX/NY/NZ768768768
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2450.378-0.000

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Supplemental data

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Additional map: unfiltered map of nucleotide-free LptB2FGC

Fileemd_9125_additional.map
Annotationunfiltered map of nucleotide-free LptB2FGC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LptB2FGC

EntireName: LptB2FGC
Components
  • Complex: LptB2FGC
    • Protein or peptide: Lipopolysaccharide export system permease protein LptF
    • Protein or peptide: Lipopolysaccharide export system permease protein LptG
    • Protein or peptide: Lipopolysaccharide export system ATP-binding protein LptB
    • Protein or peptide: Lipopolysaccharide export system protein LptC
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE

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Supramolecule #1: LptB2FGC

SupramoleculeName: LptB2FGC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12

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Macromolecule #1: Lipopolysaccharide export system permease protein LptF

MacromoleculeName: Lipopolysaccharide export system permease protein LptF
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 40.393473 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIIIRYLVRE TLKSQLAILF ILLLIFFCQK LVRILGAAVD GDIPANLVLS LLGLGVPEMA QLILPLSLFL GLLMTLGKLY TESEITVMH ACGLSKAVLV KAAMILAVFT AIVAAVNVMW AGPWSSRHQD EVLAEAKANP GMAALAQGQF QQATNGSSVL F IESVDGSD ...String:
MIIIRYLVRE TLKSQLAILF ILLLIFFCQK LVRILGAAVD GDIPANLVLS LLGLGVPEMA QLILPLSLFL GLLMTLGKLY TESEITVMH ACGLSKAVLV KAAMILAVFT AIVAAVNVMW AGPWSSRHQD EVLAEAKANP GMAALAQGQF QQATNGSSVL F IESVDGSD FKDVFLAQIR PKGNARPSVV VADSGHLTQL RDGSQVVTLN QGTRFEGTAL LRDFRITDFQ DYQAIIGHQA VA LDPNDTD QMDMRTLWNT DTDRARAELN WRITLVFTVF MMALMVVPLS VVNPRQGRVL SMLPAMLLYL LFFLIQTSLK SNG GKGKLD PTLWMWTVNL IYLALAIVLN LWDTVPVRRL RASFSRKGAV

UniProtKB: Lipopolysaccharide export system permease protein LptF

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Macromolecule #2: Lipopolysaccharide export system permease protein LptG

MacromoleculeName: Lipopolysaccharide export system permease protein LptG
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 39.65141 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQPFGVLDRY IGKTIFTTIM MTLFMLVSLS GIIKFVDQLK KAGQGSYDAL GAGMYTLLSV PKDVQIFFPM AALLGALLGL GMLAQRSEL VVMQASGFTR MQVALSVMKT AIPLVLLTMA IGEWVAPQGE QMARNYRAQA MYGGSLLSTQ QGLWAKDGNN F VYIERVKG ...String:
MQPFGVLDRY IGKTIFTTIM MTLFMLVSLS GIIKFVDQLK KAGQGSYDAL GAGMYTLLSV PKDVQIFFPM AALLGALLGL GMLAQRSEL VVMQASGFTR MQVALSVMKT AIPLVLLTMA IGEWVAPQGE QMARNYRAQA MYGGSLLSTQ QGLWAKDGNN F VYIERVKG DEELGGISIY AFNENRRLQS VRYAATAKFD PEHKVWRLSQ VDESDLTNPK QITGSQTVSG TWKTNLTPDK LG VVALDPD ALSISGLHNY VKYLKSSGQD AGRYQLNMWS KIFQPLSVAV MMLMALSFIF GPLRSVPMGV RVVTGISFGF VFY VLDQIF GPLTLVYGIP PIIGALLPSA SFFLISLWLL MRKS

UniProtKB: Lipopolysaccharide export system permease protein LptG

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Macromolecule #3: Lipopolysaccharide export system ATP-binding protein LptB

MacromoleculeName: Lipopolysaccharide export system ATP-binding protein LptB
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 28.131088 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHHH MATLTAKNLA KAYKGRRVVE DVSLTVNSGE IVGLLGPNGA GKTTTFYMVV GIVPRDAGNI IIDDDDISLL PLHARARRG IGYLPQEASI FRRLSVYDNL MAVLQIRDDL SAEQREDRAN ELMEEFHIEH LRDSMGQSLS GGERRRVEIA R ALAANPKF ...String:
MGHHHHHHHH MATLTAKNLA KAYKGRRVVE DVSLTVNSGE IVGLLGPNGA GKTTTFYMVV GIVPRDAGNI IIDDDDISLL PLHARARRG IGYLPQEASI FRRLSVYDNL MAVLQIRDDL SAEQREDRAN ELMEEFHIEH LRDSMGQSLS GGERRRVEIA R ALAANPKF ILLDEPFAGV DPISVIDIKR IIEHLRDSGL GVLITDHNVR ETLAVCERAY IVSQGHLIAH GTPTEILQDE HV KRVYLGE DFRL

UniProtKB: Lipopolysaccharide export system ATP-binding protein LptB

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Macromolecule #4: Lipopolysaccharide export system protein LptC

MacromoleculeName: Lipopolysaccharide export system protein LptC / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 23.808877 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MWSHPQFEKE NLYFQGGMSK ARRWVIIVLS LAVLVMIGIN MAEKDDTAQV VVNNNDPTYK SEHTDTLVYN PEGALSYRLI AQHVEYYSD QAVSWFTQPV LTTFDKDKIP TWSVKADKAK LTNDRMLYLY GHVEVNALVP DSQLRRITTD NAQINLVTQD V TSEDLVTL ...String:
MWSHPQFEKE NLYFQGGMSK ARRWVIIVLS LAVLVMIGIN MAEKDDTAQV VVNNNDPTYK SEHTDTLVYN PEGALSYRLI AQHVEYYSD QAVSWFTQPV LTTFDKDKIP TWSVKADKAK LTNDRMLYLY GHVEVNALVP DSQLRRITTD NAQINLVTQD V TSEDLVTL YGTTFNSSGL KMRGNLRSKN AELIEKVRTS YEIQNKQTQP

UniProtKB: Lipopolysaccharide export system protein LptC

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Macromolecule #5: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 5 / Number of copies: 1 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM

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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

Concentration4 mg/mL
BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE
Detailsnanodisc incorporated LptB2FGC

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 213774
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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