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- EMDB-11555: Cryo-EM structure of MlaFEDB in complex with AMP-PNP -

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Basic information

Entry
Database: EMDB / ID: EMD-11555
TitleCryo-EM structure of MlaFEDB in complex with AMP-PNP
Map data
Sample
  • Complex: MlaFEDB
    • Complex: YrbD protein
      • Protein or peptide: YrbD protein
    • Complex: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
      • Protein or peptide: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
    • Complex: Toluene tolerance protein Ttg2A
      • Protein or peptide: Toluene tolerance protein Ttg2A
    • Complex: Uncharacterized protein
      • Protein or peptide: Uncharacterized protein
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Keywordsphospholipid / phospholipid transport / ABC transporter / MlaFEDB / MlaFE / MlaD / MlaE / MlaF / MlaB / outer membrane / Mla transport pathway / LIPID TRANSPORT
Function / homology
Function and homology information


phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid-translocating ATPase complex / phospholipid transport / ATP-binding cassette (ABC) transporter complex / phospholipid binding / response to antibiotic / DNA damage response / ATP hydrolysis activity / ATP binding ...phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid-translocating ATPase complex / phospholipid transport / ATP-binding cassette (ABC) transporter complex / phospholipid binding / response to antibiotic / DNA damage response / ATP hydrolysis activity / ATP binding / membrane / plasma membrane / cytosol
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 ...: / : / 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
YrbD protein / Intermembrane phospholipid transport system permease protein MlaE / Intermembrane phospholipid transport system binding protein MlaB / Intermembrane phospholipid transport system binding protein MlaD / Toluene tolerance protein Ttg2A / Anti-sigma factor antagonist
Similarity search - Component
Biological speciesEscherichia coli B185 (bacteria) / Escherichia coli (E. coli) / Escherichia coli 909945-2 (bacteria) / Escherichia coli 2.3916 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDong CJ / Dong HH
Funding support China, United Kingdom, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)2017YFA0504803, 2018YFA0507700, 2017YFC0840100, 2017YFC00840101,31900039,81971974 China
Wellcome TrustWT106121MA United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB.
Authors: Xiaodi Tang / Shenghai Chang / Wen Qiao / Qinghua Luo / Yuejia Chen / Zhiying Jia / James Coleman / Ke Zhang / Ting Wang / Zhibo Zhang / Changbin Zhang / Xiaofeng Zhu / Xiawei Wei / ...Authors: Xiaodi Tang / Shenghai Chang / Wen Qiao / Qinghua Luo / Yuejia Chen / Zhiying Jia / James Coleman / Ke Zhang / Ting Wang / Zhibo Zhang / Changbin Zhang / Xiaofeng Zhu / Xiawei Wei / Changjiang Dong / Xing Zhang / Haohao Dong /
Abstract: The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by ...The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3-4.1 Å and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA-OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development.
History
DepositionJul 30, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zy9
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11555.png

Downloads & links

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Map

FileDownload / File: emd_11555.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 259.584 Å		1.01 Å/pix.
x 256 pix.
= 259.584 Å		1.01 Å/pix.
x 256 pix.
= 259.584 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.014 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.10341957 - 0.16468902
Average (Standard dev.)0.00026394738 (±0.0039293077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 259.584 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z259.584259.584259.584
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1030.1650.000

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Supplemental data

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Sample components

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Entire : MlaFEDB

EntireName: MlaFEDB
Components
  • Complex: MlaFEDB
    • Complex: YrbD protein
      • Protein or peptide: YrbD protein
    • Complex: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
      • Protein or peptide: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
    • Complex: Toluene tolerance protein Ttg2A
      • Protein or peptide: Toluene tolerance protein Ttg2A
    • Complex: Uncharacterized protein
      • Protein or peptide: Uncharacterized protein
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: MlaFEDB

SupramoleculeName: MlaFEDB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: YrbD protein

SupramoleculeName: YrbD protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli B185 (bacteria)

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Supramolecule #3: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS ...

SupramoleculeName: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #4: Toluene tolerance protein Ttg2A

SupramoleculeName: Toluene tolerance protein Ttg2A / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Escherichia coli 909945-2 (bacteria)

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Supramolecule #5: Uncharacterized protein

SupramoleculeName: Uncharacterized protein / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli 2.3916 (bacteria)

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Macromolecule #1: YrbD protein

MacromoleculeName: YrbD protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli B185 (bacteria)
Molecular weightTheoretical: 19.593133 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MQTKKNEIWV GIFLLAALLA ALFVCLKAAN VTSIRTEPTY TLYATFDNIG GLKARSPVSI GGVVVGRVAD ITLDPKTYLP RVTLEIEQR YNHIPDTSSL SIRTSGLLGE QYLALNVGFE DPELGTAILK DGDTIQDTKS AMVLEDLIGQ FLYGSKGDDN K NSGDAPAA APGNNETTEP VGTTK

UniProtKB: YrbD protein

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Macromolecule #2: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS ...

MacromoleculeName: ABC transporter maintaining OM lipid asymmetry, cytoplasmic STAS component
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.795488 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MSESLSWMQT GDTLALSGEL DQDVLLPLWE MREEAVKGIT CIDLSRVSRV DTGGLALLLH LIDLAKKQGN NVTLQGVNDK VYTLAKLYN LPADVLPRHH HHHHHH

UniProtKB: Anti-sigma factor antagonist

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Macromolecule #3: Toluene tolerance protein Ttg2A

MacromoleculeName: Toluene tolerance protein Ttg2A / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli 909945-2 (bacteria)
Molecular weightTheoretical: 29.128801 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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: Toluene tolerance protein Ttg2A

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Macromolecule #4: Uncharacterized protein

MacromoleculeName: Uncharacterized protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli 2.3916 (bacteria)
Molecular weightTheoretical: 27.885162 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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 #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / 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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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.8
Component:
ConcentrationFormula
20.0 mMTris-HCl
150.0 mMNaCl
0.05 %LMNG

Details: 20 mM Tris-Cl, pH 7.8, 150 mM NaCl and 0.05% LMNG
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 793723
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 133480
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-6zy9:
Cryo-EM structure of MlaFEDB in complex with AMP-PNP

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