+Open data
-Basic information
Entry | Database: PDB / ID: 7ch6 | ||||||
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Title | Cryo-EM structure of E.coli MlaFEB with AMPPNP | ||||||
Components |
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Keywords | MEMBRANE 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 | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
Authors | Zhou, C. / Shi, H. / Zhang, M. / Huang, Y. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ch6.cif.gz | 209.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ch6.ent.gz | 168.5 KB | Display | PDB format |
PDBx/mmJSON format | 7ch6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ch/7ch6 ftp://data.pdbj.org/pub/pdb/validation_reports/ch/7ch6 | HTTPS FTP |
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-Related structure data
Related structure data | 30369MC 7ch7C 7ch8C 7ch9C 7chaC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 K12 (bacteria) / References: UniProt: A0A4S5B3V0 #2: Protein | 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 K12 (bacteria) / References: UniProt: A0A4V3YUQ9 #3: Protein | 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 K12 (bacteria) / References: UniProt: A0A4S5B5E3 #4: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: E.coli MlaFEB bond with AMPPNP / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Source (natural) | Organism: Escherichia coli K12 (bacteria) |
Source (recombinant) | Organism: Escherichia coli K12 (bacteria) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.17.1_3660: / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115430 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||
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