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- EMDB-30371: Cryo-EM structure of P.aeruginosa MlaFEBD with ADP-V -

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Basic information

Entry
Database: EMDB / ID: EMD-30371
TitleCryo-EM structure of P.aeruginosa MlaFEBD with ADP-V
Map data
Sample
  • Complex: E.coli MlaFEB bond with AMPPNP
    • Protein or peptide: MlaD domain-containing protein
    • Protein or peptide: Probable permease of ABC transporter
    • Protein or peptide: Probable ATP-binding component of ABC transporter
    • Protein or peptide: STAS domain-containing protein
  • Ligand: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
  • Ligand: ADP METAVANADATE
  • Ligand: MAGNESIUM ION
KeywordsMla complex / Lipid transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


response to nitrite / phospholipid transporter activity / phospholipid transport / ATP-binding cassette (ABC) transporter complex / phospholipid binding / ATP hydrolysis activity / extracellular region / ATP binding / plasma membrane
Similarity search - Function
Probable phospholipid ABC transporter-binding protein MlaD / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / Mce/MlaD / MlaD protein / STAS domain profile. / STAS domain / STAS domain superfamily ...Probable phospholipid ABC transporter-binding protein MlaD / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / Mce/MlaD / MlaD protein / STAS domain profile. / STAS domain / STAS domain superfamily / 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
Probable ATP-binding component of ABC transporter / Intermembrane phospholipid transport system permease protein MlaE / Mce/MlaD domain-containing protein / STAS domain-containing protein
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZhou C / Shi H
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Mol Biol / Year: 2021
Title: Structural Insight into Phospholipid Transport by the MlaFEBD Complex from P. aeruginosa.
Authors: Changping Zhou / Huigang Shi / Manfeng Zhang / Lijun Zhou / Le Xiao / Shasha Feng / Wonpil Im / Min Zhou / Xinzheng Zhang / Yihua Huang /
Abstract: The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic ...The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaFEBD, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.
History
DepositionJul 5, 2020-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ch8
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30371.png

Downloads & links

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Map

FileDownload / File: emd_30371.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0238 / Movie #1: 0.018
Minimum - Maximum-0.048475426 - 0.09980438
Average (Standard dev.)0.00003490441 (±0.0030274852)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0480.1000.000

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Supplemental data

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Sample components

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Entire : E.coli MlaFEB bond with AMPPNP

EntireName: E.coli MlaFEB bond with AMPPNP
Components
  • Complex: E.coli MlaFEB bond with AMPPNP
    • Protein or peptide: MlaD domain-containing protein
    • Protein or peptide: Probable permease of ABC transporter
    • Protein or peptide: Probable ATP-binding component of ABC transporter
    • Protein or peptide: STAS domain-containing protein
  • Ligand: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
  • Ligand: ADP METAVANADATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: E.coli MlaFEB bond with AMPPNP

SupramoleculeName: E.coli MlaFEB bond with AMPPNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: MlaD domain-containing protein

MacromoleculeName: MlaD domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 16.571945 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
MQTRTLEIGV GLFLLAGLLA LLLLALRVSG LSVGNAGDTY KVYAYFDNIA GVTVRGKVTL AGVTIGKVTA VDLDRDSYTG RVTMEINQN VNNLPVDSTA SILTAGLLGE KYIGISVGGD EDVLKDGSTI HDTQSALVLE DLIGKFLLNS VNKDEAKK

UniProtKB: Mce/MlaD domain-containing protein

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Macromolecule #2: Probable permease of ABC transporter

MacromoleculeName: Probable permease of ABC transporter / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 28.400326 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MRRVSPLERI RLFGRAGLDV VAALGRSTLF LGHALLGRRT PGTGLHLLVK QLYSVGVLSL AIIVVSGLFI GMVLALQGYN ILISYGSEQ AVGQMVALTL LRELGPVVTG LLFAGRAGSA LTAEIGNMKA TEQLSSLEMI GVDPLKYIVA PRLWAGFISM P LLAAIFSV ...String:
MRRVSPLERI RLFGRAGLDV VAALGRSTLF LGHALLGRRT PGTGLHLLVK QLYSVGVLSL AIIVVSGLFI GMVLALQGYN ILISYGSEQ AVGQMVALTL LRELGPVVTG LLFAGRAGSA LTAEIGNMKA TEQLSSLEMI GVDPLKYIVA PRLWAGFISM P LLAAIFSV VGIWGGAMVA VDWLGVYEGS FWANMQNSVQ FTEDVLNGVI KSIVFAFVVT WIAVYQGYDC EPTSEGISRA TT RTVVYAS LAVLGLDFIL TALMFGDF

UniProtKB: Intermembrane phospholipid transport system permease protein MlaE

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Macromolecule #3: Probable ATP-binding component of ABC transporter

MacromoleculeName: Probable ATP-binding component of ABC transporter / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 29.657367 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MSTDSAYAVE LKGLTFKRGS RAIFDNIDVR IPRGKVTGIM GPSGCGKTTL LRLIASQLRP SKGEVWVNGQ NLPQLSRGDL FDMRKQFGV LFQSGALFTD LDVFENVAFP LRVHTQLPEE MIRDIVLMKL QAVGLRGAVE LMPDELSGGM KRRVALARAI A LDPQILLY ...String:
MSTDSAYAVE LKGLTFKRGS RAIFDNIDVR IPRGKVTGIM GPSGCGKTTL LRLIASQLRP SKGEVWVNGQ NLPQLSRGDL FDMRKQFGV LFQSGALFTD LDVFENVAFP LRVHTQLPEE MIRDIVLMKL QAVGLRGAVE LMPDELSGGM KRRVALARAI A LDPQILLY DEPFVGQDPI AMGVLVRLIR LLNDALGITS IVVSHDLAET ASIADYIYIV GDGRVLGHGT PDVLKETDDP RI RQFVKGI PDGPVPFHYP ARDYRADLLG ER

UniProtKB: Probable ATP-binding component of ABC transporter

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Macromolecule #4: STAS domain-containing protein

MacromoleculeName: STAS domain-containing protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 10.789388 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
MSQASLREGA AGELQLAGVL DYSSGPALRE QGGRLIRASQ AAELVVDCSA VERSSSVGIS LLLAFIRDAR KAGKVLSVRA LPDDMREIA KVSSLLEILP LQE

UniProtKB: STAS domain-containing protein

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Macromolecule #5: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE

MacromoleculeName: 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE
type: ligand / ID: 5 / Number of copies: 8 / Formula: LPP
Molecular weightTheoretical: 648.891 Da
Chemical component information

ChemComp-LPP:
2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE / phospholipid*YM

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Macromolecule #6: ADP METAVANADATE

MacromoleculeName: ADP METAVANADATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: AD9
Molecular weightTheoretical: 527.149 Da
Chemical component information

ChemComp-AD9:
ADP METAVANADATE

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: DIFFRACTION / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 560624
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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