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- EMDB-30367: Cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformat... -

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Basic information

Entry
Database: EMDB / ID: EMD-30367
TitleCryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
Map datacryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
Sample
  • Complex: cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
    • Protein or peptide: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
    • Protein or peptide: Phospholipid ABC transporter ATP-binding protein MlaF
    • Protein or peptide: Lipid asymmetry maintenance protein MlaB
    • Protein or peptide: Outer membrane lipid asymmetry maintenance protein MlaD
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid transporter activity / phospholipid-translocating ATPase complex / phospholipid transport / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ATPase-coupled transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / phospholipid binding / response to antibiotic ...phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid transporter activity / phospholipid-translocating ATPase complex / phospholipid transport / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ATPase-coupled transmembrane transporter activity / 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 ABC transporter ATP-binding protein MlaF/Mkl / : / : / : / Probable phospholipid ABC transporter-binding protein MlaD / ABC transport permease subunit MlaE, Proteobacteria / : / ABC transporter permease MalE / Permease MlaE / STAS domain ...Probable ABC transporter ATP-binding protein MlaF/Mkl / : / : / : / 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
Intermembrane phospholipid transport system permease protein MlaE / Lipid asymmetry maintenance protein MlaB / Phospholipid ABC transporter ATP-binding protein MlaF / Outer membrane lipid asymmetry maintenance protein MlaD / Intermembrane phospholipid transport system ATP-binding protein MlaF / Intermembrane phospholipid transport system binding protein MlaB / Intermembrane phospholipid transport system binding protein MlaD / Intermembrane phospholipid transport system permease protein MlaE
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsChi XM / Fan QX
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31930059 China
CitationJournal: Cell Res / Year: 2020
Title: Structural mechanism of phospholipids translocation by MlaFEDB complex.
Authors: Ximin Chi / Qiongxuan Fan / Yuanyuan Zhang / Ke Liang / Li Wan / Qiang Zhou / Yanyan Li /
Abstract: In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier ...In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier to exclude the entry of most antibiotics. Understanding the mechanisms of phospholipid translocation between the inner and outer membrane represents one of the major challenges surrounding bacterial phospholipid homeostasis. The conserved MlaFEDB complex in the inner membrane functions as an ABC transporter to drive the translocation of phospholipids between the inner membrane and the periplasmic protein MlaC. However, the mechanism of phospholipid translocation remains elusive. Here we determined three cryo-EM structures of MlaFEDB from Escherichia coli in its nucleotide-free and ATP-bound conformations, and performed extensive functional studies to verify and extend our findings from structural analyses. Our work reveals unique structural features of the entire MlaFEDB complex, six well-resolved phospholipids in three distinct cavities, and large-scale conformational changes upon ATP binding. Together, these findings define the cycle of structural rearrangement of MlaFEDB in action, and suggest that MlaFEDB uses an extrusion mechanism to extract and release phospholipids through the central translocation cavity.
History
DepositionJul 3, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ch0
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30367.png

Downloads & links

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Map

FileDownload / File: emd_30367.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 200 pix.
= 217.4 Å		1.09 Å/pix.
x 200 pix.
= 217.4 Å		1.09 Å/pix.
x 200 pix.
= 217.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.087 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.111392185 - 0.18417917
Average (Standard dev.)0.00070422405 (±0.009699256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 217.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0871.0871.087
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z217.400217.400217.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1110.1840.001

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Supplemental data

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Sample components

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Entire : cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformat...

EntireName: cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
Components
  • Complex: cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
    • Protein or peptide: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
    • Protein or peptide: Phospholipid ABC transporter ATP-binding protein MlaF
    • Protein or peptide: Lipid asymmetry maintenance protein MlaB
    • Protein or peptide: Outer membrane lipid asymmetry maintenance protein MlaD
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformat...

SupramoleculeName: cryo EM map of the MlaFEDB complex in ATP-bound EQclose conformation (Mutation of E170Q on MlaF)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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

MacromoleculeName: Lipid asymmetry maintenance ABC transporter permease subunit MlaE
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 27.885162 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (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 #2: Phospholipid ABC transporter ATP-binding protein MlaF

MacromoleculeName: Phospholipid ABC transporter ATP-binding protein MlaF / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 29.127816 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (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 DQPFVGQDPI TMGVLVKLIS ELNSALGVTC VVVSHDVPEV LSIADHAWIL ADKKIVAHGS AQALQANPDP RV RQFLDGI ADGPVPFRYP AGDYHADLLP GS

UniProtKB: Phospholipid ABC transporter ATP-binding protein MlaF

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Macromolecule #3: Lipid asymmetry maintenance protein MlaB

MacromoleculeName: Lipid asymmetry maintenance protein MlaB / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 10.690313 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
MSESLSWMQT GDTLALSGEL DQDVLLPLWE MREEAVKGIT CIDLSRVSRV DTGGLALLLH LIDLAKKQGN NVTLQGVNDK VYTLAKLYN LPADVLPR

UniProtKB: Lipid asymmetry maintenance protein MlaB

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Macromolecule #4: Outer membrane lipid asymmetry maintenance protein MlaD

MacromoleculeName: Outer membrane lipid asymmetry maintenance protein MlaD
type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 19.593133 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
MQTKKNEIWV GIFLLAALLA ALFVCLKAAN VTSIRTEPTY TLYATFDNIG GLKARSPVSI GGVVVGRVAD ITLDPKTYLP RVTLEIEQR YNHIPDTSSL SIRTSGLLGE QYLALNVGFE DPELGTAILK DGDTIQDTKS AMVLEDLIGQ FLYGSKGDDN K NSGDAPAA APGNNETTEP VGTTK

UniProtKB: Outer membrane lipid asymmetry maintenance protein MlaD

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 21159
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD

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