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- PDB-7ch7: Cryo-EM structure of E.coli MlaFEB -

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Basic information

Entry
Database: PDB / ID: 7ch7
TitleCryo-EM structure of E.coli MlaFEB
Components
  • Lipid asymmetry maintenance ABC transporter permease subunit MlaE
  • Lipid asymmetry maintenance protein MlaB
  • Phospholipid ABC transporter ATP-binding protein MlaF
KeywordsMEMBRANE PROTEIN / Mla complex / Lipid transporter
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 / Transcription Regulator spoIIAA / STAS domain profile. / STAS domain / STAS domain superfamily ...Probable ABC transporter ATP-binding protein MlaF/Mkl / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / STAS domain / Transcription Regulator spoIIAA / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
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 (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZhou, C. / Shi, H. / Huang, Y.
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 19, 2021Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 19, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 26, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Jul 2, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
B: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
C: Phospholipid ABC transporter ATP-binding protein MlaF
D: Phospholipid ABC transporter ATP-binding protein MlaF
E: Lipid asymmetry maintenance protein MlaB
F: Lipid asymmetry maintenance protein MlaB


Theoretical massNumber of molelcules
Total (without water)135,4096
Polymers135,4096
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15030 Å2
ΔGint-122 kcal/mol
Surface area46560 Å2

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Components

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


Mass: 27885.162 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mlaE, FAZ83_04790 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A4S5B3V0
#2: Protein Phospholipid ABC transporter ATP-binding protein MlaF


Mass: 29128.801 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mlaF, FAZ83_04795 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A4V3YUQ9
#3: Protein Lipid asymmetry maintenance protein MlaB


Mass: 10690.313 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mlaB, FAZ83_04775 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A4S5B5E3
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E.coli Mla FEB complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: YES
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFIND4.1CTF correction
8PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74479 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0079370
ELECTRON MICROSCOPYf_angle_d0.85212730
ELECTRON MICROSCOPYf_dihedral_angle_d23.463392
ELECTRON MICROSCOPYf_chiral_restr0.0471528
ELECTRON MICROSCOPYf_plane_restr0.0061604

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