+Open data
-Basic information
Entry | Database: PDB / ID: 7ch8 | ||||||
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Title | Cryo-EM structure of P.aeruginosa MlaFEBD with ADP-V | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / Mla complex / Lipid transporter | ||||||
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 | ||||||
Biological species | Pseudomonas aeruginosa (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Zhou, C. / Shi, H. / Zhang, M. / Huang, Y. | ||||||
Funding support | China, 1items
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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 | 7ch8.cif.gz | 360.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ch8.ent.gz | 296.3 KB | Display | PDB format |
PDBx/mmJSON format | 7ch8.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ch8_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 7ch8_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7ch8_validation.xml.gz | 78.6 KB | Display | |
Data in CIF | 7ch8_validation.cif.gz | 111.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ch/7ch8 ftp://data.pdbj.org/pub/pdb/validation_reports/ch/7ch8 | HTTPS FTP |
-Related structure data
Related structure data | 30371MC 7ch6C 7ch7C 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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1 |
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-Components
-Protein , 4 types, 12 molecules ABCDEFGHIJKL
#1: Protein | Mass: 16571.945 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) 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 Gene: PA4454 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q9HVW3 #2: Protein | Mass: 28400.326 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) 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 Gene: PA4455 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q9HVW2 #3: Protein | Mass: 29657.367 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) 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 Gene: PA4456 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q9HVW1 #4: Protein | Mass: 10789.388 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) 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 Gene: PA4452 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q9HVW5 |
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-Non-polymers , 3 types, 12 molecules
#5: Chemical | ChemComp-LPP / #6: Chemical | #7: Chemical | |
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-Details
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-#4 / Source: RECOMBINANT |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Source (recombinant) | Organism: Escherichia coli K-12 (bacteria) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
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 / 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 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 560624 / Symmetry type: POINT | ||||||||||||||||||||||||
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