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- EMDB-16904: Structure of the MlaCD complex (1:6 stoichiometry) -

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Basic information

Entry
Database: EMDB / ID: EMD-16904
TitleStructure of the MlaCD complex (1:6 stoichiometry)
Map data
Sample
  • Complex: MlaCD
    • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaC
    • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaD
KeywordsOuter membrane / gram-negative bacteria / lipid transfer / antibiotic resistance / LIPID BINDING PROTEIN
Function / homology
Function and homology information


phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid transporter activity / phospholipid-translocating ATPase complex / phospholipid transport / phospholipid binding / outer membrane-bounded periplasmic space / membrane / plasma membrane
Similarity search - Function
Tgt2/MlaC superfamily / Toluene tolerance Ttg2/phospholipid-binding protein MlaC / MlaC protein / Probable phospholipid ABC transporter-binding protein MlaD / : / Mce/MlaD / MlaD protein
Similarity search - Domain/homology
Intermembrane phospholipid transport system binding protein MlaC / Intermembrane phospholipid transport system binding protein MlaD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.35 Å
AuthorsWotherspoon P / Bui S / Sridhar P / Bergeron JRC / Knowles TJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R019061/2 United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport.
Authors: Peter Wotherspoon / Hannah Johnston / David J Hardy / Rachel Holyfield / Soi Bui / Giedrė Ratkevičiūtė / Pooja Sridhar / Jonathan Colburn / Charlotte B Wilson / Adam Colyer / Benjamin F ...Authors: Peter Wotherspoon / Hannah Johnston / David J Hardy / Rachel Holyfield / Soi Bui / Giedrė Ratkevičiūtė / Pooja Sridhar / Jonathan Colburn / Charlotte B Wilson / Adam Colyer / Benjamin F Cooper / Jack A Bryant / Gareth W Hughes / Phillip J Stansfeld / Julien R C Bergeron / Timothy J Knowles /
Abstract: The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane ...The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
History
DepositionMar 23, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16904.png

Downloads & links

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Map

FileDownload / File: emd_16904.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.71 Å/pix.
x 350 pix.
= 248.5 Å		0.71 Å/pix.
x 350 pix.
= 248.5 Å		0.71 Å/pix.
x 350 pix.
= 248.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.71 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.28342614 - 0.4986564
Average (Standard dev.)0.0014733393 (±0.012819843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 248.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16904_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16904_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MlaCD

EntireName: MlaCD
Components
  • Complex: MlaCD
    • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaC
    • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaD

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Supramolecule #1: MlaCD

SupramoleculeName: MlaCD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Intermembrane phospholipid transport system binding protein MlaC

MacromoleculeName: Intermembrane phospholipid transport system binding protein MlaC
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.989559 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFKRLMMVAL LVIAPLSAAT AADQTNPYKL MDEAAQKTFD RLKNEQPQIR ANPDYLRTIV DQELLPYVQV KYAGALVLGQ YYKSATPAQ REAYFAAFRE YLKQAYGQAL AMYHGQTYQI APEQPLGDKT IVPIRVTIID PNGRPPVRLD FQWRKNSQTG N WQAYDMIA ...String:
MFKRLMMVAL LVIAPLSAAT AADQTNPYKL MDEAAQKTFD RLKNEQPQIR ANPDYLRTIV DQELLPYVQV KYAGALVLGQ YYKSATPAQ REAYFAAFRE YLKQAYGQAL AMYHGQTYQI APEQPLGDKT IVPIRVTIID PNGRPPVRLD FQWRKNSQTG N WQAYDMIA EGVSMITTKQ NEWGTLLRTK GIDGLTAQLK SISQQKITLE EKK

UniProtKB: Intermembrane phospholipid transport system binding protein MlaC

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Macromolecule #2: Intermembrane phospholipid transport system binding protein MlaD

MacromoleculeName: Intermembrane phospholipid transport system binding protein MlaD
type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 19.593133 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQTKKNEIWV GIFLLAALLA ALFVCLKAAN VTSIRTEPTY TLYATFDNIG GLKARSPVSI GGVVVGRVAD ITLDPKTYLP RVTLEIEQR YNHIPDTSSL SIRTSGLLGE QYLALNVGFE DPELGTAILK DGDTIQDTKS AMVLEDLIGQ FLYGSKGDDN K NSGDAPAA APGNNETTEP VGTTK

UniProtKB: Intermembrane phospholipid transport system binding protein MlaD

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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 (6k 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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97460
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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