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- PDB-7ch8: Cryo-EM structure of P.aeruginosa MlaFEBD with ADP-V -

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Basic information

Entry
Database: PDB / ID: 7ch8
TitleCryo-EM structure of P.aeruginosa MlaFEBD with ADP-V
Components
  • MlaD domain-containing protein
  • Probable ATP-binding component of ABC transporter
  • Probable permease of ABC transporter
  • STAS domain-containing protein
KeywordsMEMBRANE 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
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
ADP METAVANADATE / Chem-LPP / 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 speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZhou, C. / Shi, H. / Zhang, M. / Huang, Y.
Funding support China, 1items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: MlaD domain-containing protein
B: MlaD domain-containing protein
C: MlaD domain-containing protein
D: MlaD domain-containing protein
E: MlaD domain-containing protein
F: MlaD domain-containing protein
G: Probable permease of ABC transporter
H: Probable permease of ABC transporter
I: Probable ATP-binding component of ABC transporter
J: Probable ATP-binding component of ABC transporter
K: STAS domain-containing protein
L: STAS domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,42024
Polymers237,12612
Non-polymers6,29412
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 12 molecules ABCDEFGHIJKL

#1: Protein
MlaD domain-containing 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 Probable permease of ABC transporter


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 Probable ATP-binding component of ABC transporter


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 STAS domain-containing 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 / 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE / 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / L-B,G-DIPALMITOYL-A-PHOSPHATIDIC ACID DISODIUM SALT / 3-SN-PHOSPHATIDIC ACID / 1,2-DIPALMITOYLDISODIUM SALT


Mass: 648.891 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C35H69O8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#6: Chemical ChemComp-AD9 / ADP METAVANADATE


Mass: 527.149 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P2V / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY

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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 MlaFEB bond with AMPPNP / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / 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
4CTFFIND4.1CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 560624 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416545
ELECTRON MICROSCOPYf_angle_d0.69122388
ELECTRON MICROSCOPYf_dihedral_angle_d27.5172538
ELECTRON MICROSCOPYf_chiral_restr0.0442701
ELECTRON MICROSCOPYf_plane_restr0.0042793

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