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- EMDB-30369: Cryo-EM structure of E.coli MlaFEB with AMPPNP -

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Basic information

Entry
Database: EMDB / ID: EMD-30369
TitleCryo-EM structure of E.coli MlaFEB with AMPPNP
Map data
Sample
  • Complex: E.coli MlaFEB bond with AMPPNP
    • Protein or peptide: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
    • Protein or peptide: Phospholipid ABC transporter ATP-binding protein MlaF
    • Protein or peptide: Lipid asymmetry maintenance protein MlaB
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsMla complex / Lipid transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


ATPase-coupled transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / ATP binding
Similarity search - Function
Probable ABC transporter ATP-binding protein MlaF/Mkl / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / STAS domain profile. / STAS domain / STAS domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. ...Probable ABC transporter ATP-binding protein MlaF/Mkl / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / 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
Intermembrane phospholipid transport system permease protein MlaE / Lipid asymmetry maintenance protein MlaB / Phospholipid ABC transporter ATP-binding protein MlaF
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhou C / Shi H
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) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ch6
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30369.png

Downloads & links

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Map

FileDownload / File: emd_30369.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.0361 / Movie #1: 0.0361
Minimum - Maximum-0.09027442 - 0.17398834
Average (Standard dev.)0.00009483083 (±0.004677198)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0900.1740.000

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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: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
    • Protein or peptide: Phospholipid ABC transporter ATP-binding protein MlaF
    • Protein or peptide: Lipid asymmetry maintenance protein MlaB
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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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-#3
Source (natural)Organism: Escherichia coli K12 (bacteria)

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Macromolecule #1: Lipid asymmetry maintenance ABC transporter permease subunit MlaE

MacromoleculeName: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 27.885162 KDa
Recombinant expressionOrganism: Escherichia coli K12 (bacteria)
SequenceString: MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGM LVALSLLREL GPVVAALLFA GRAGSALTAE IGLMRATEQL SSMEMMAVDP LRRVISPRFW AGVISLPLLT V IFVAVGIW ...String:
MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGM LVALSLLREL GPVVAALLFA GRAGSALTAE IGLMRATEQL SSMEMMAVDP LRRVISPRFW AGVISLPLLT V IFVAVGIW GGSLVGVSWK GIDSGFFWSA MQNAVDWRMD LVNCLIKSVV FAITVTWISL FNGYDAIPTS AGISRATTRT VV HSSLAVL GLDFVLTALM FGN

UniProtKB: Intermembrane phospholipid transport system permease protein MlaE

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Macromolecule #2: Phospholipid ABC transporter ATP-binding protein MlaF

MacromoleculeName: Phospholipid ABC transporter ATP-binding protein MlaF / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 29.128801 KDa
Recombinant expressionOrganism: Escherichia coli K12 (bacteria)
SequenceString: MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSM LFQSGALFTD MNVFDNVAYP LREHTQLPAP LLHSTVMMKL EAVGLRGAAK LMPSELSGGM ARRAALARAI A LEPDLIMF ...String:
MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSM LFQSGALFTD MNVFDNVAYP LREHTQLPAP LLHSTVMMKL EAVGLRGAAK LMPSELSGGM ARRAALARAI A LEPDLIMF DEPFVGQDPI TMGVLVKLIS ELNSALGVTC VVVSHDVPEV LSIADHAWIL ADKKIVAHGS AQALQANPDP RV RQFLDGI ADGPVPFRYP AGDYHADLLP GS

UniProtKB: Phospholipid ABC transporter ATP-binding protein MlaF

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Macromolecule #3: Lipid asymmetry maintenance protein MlaB

MacromoleculeName: Lipid asymmetry maintenance protein MlaB / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 10.690313 KDa
Recombinant expressionOrganism: Escherichia coli K12 (bacteria)
SequenceString:
MSESLSWMQT GDTLALSGEL DQDVLLPLWE MREEAVKGIT CIDLSRVSRV DTGGLALLLH LIDLAKKQGN NVTLQGVNDK VYTLAKLYN LPADVLPR

UniProtKB: Lipid asymmetry maintenance protein MlaB

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 115430
FSC plot (resolution estimation)

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