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- EMDB-40162: E. coli MlaC bound to MlaFEDB -

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Basic information

Entry
Database: EMDB / ID: EMD-40162
TitleE. coli MlaC bound to MlaFEDB
Map dataE. coli MlaFEDB bound to MlaC, Map 1
Sample
  • Complex: E. coli MlaC bound to MlaFEDB
    • Protein or peptide: MlaE
    • Protein or peptide: MlaF
    • Protein or peptide: MlaD
    • Protein or peptide: MlaB
    • Protein or peptide: Foldon-MlaC
KeywordsE. coli / bacteria / outer membrane / cryo-EM / lipid transport / mla / structural biology
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsMacRae MR / Coudray N / Ekiert D / Bhabha G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128777 United States
CitationJournal: J Biol Chem / Year: 2023
Title: Protein-protein interactions in the Mla lipid transport system probed by computational structure prediction and deep mutational scanning.
Authors: Mark R MacRae / Dhenesh Puvanendran / Max A B Haase / Nicolas Coudray / Ljuvica Kolich / Cherry Lam / Minkyung Baek / Gira Bhabha / Damian C Ekiert /
Abstract: The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from external stressors, such as antibiotics. The Mla transport system is implicated in the ...The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from external stressors, such as antibiotics. The Mla transport system is implicated in the Maintenance of OM Lipid Asymmetry by mediating retrograde phospholipid transport across the cell envelope. Mla uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein-protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC from Escherichia coli, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein-protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the bacterial inner and OMs.
History
DepositionMar 16, 2023-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40162.png

Downloads & links

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Map

FileDownload / File: emd_40162.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli MlaFEDB bound to MlaC, Map 1
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.34261838 - 0.68565667
Average (Standard dev.)-0.000030368716 (±0.034457665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 333.184 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40162_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: E. coli MlaFEDB bound to MlaC, Map 2

Fileemd_40162_additional_1.map
AnnotationE. coli MlaFEDB bound to MlaC, Map 2
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: E. coli MlaFEDB bound to MlaC, Map 3

Fileemd_40162_additional_2.map
AnnotationE. coli MlaFEDB bound to MlaC, Map 3
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: E. coli MlaFEDB bound to MlaC, Map 4

Fileemd_40162_additional_3.map
AnnotationE. coli MlaFEDB bound to MlaC, Map 4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: E. coli MlaFEDB bound to MlaC, half map A of Map 1

Fileemd_40162_half_map_1.map
AnnotationE. coli MlaFEDB bound to MlaC, half map A of Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: E. coli MlaFEDB bound to MlaC, half map B of Map 1

Fileemd_40162_half_map_2.map
AnnotationE. coli MlaFEDB bound to MlaC, half map B of Map 1
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli MlaC bound to MlaFEDB

EntireName: E. coli MlaC bound to MlaFEDB
Components
  • Complex: E. coli MlaC bound to MlaFEDB
    • Protein or peptide: MlaE
    • Protein or peptide: MlaF
    • Protein or peptide: MlaD
    • Protein or peptide: MlaB
    • Protein or peptide: Foldon-MlaC

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Supramolecule #1: E. coli MlaC bound to MlaFEDB

SupramoleculeName: E. coli MlaC bound to MlaFEDB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: MlaE

MacromoleculeName: MlaE / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGML VALSLLRELG PVVAALLFAG RAGSALTAEI GLMRATEQLS SMEMMAVDPL RRVISPRFWA GVISLPLLTV IFVAVGIWGG ...String:
MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGML VALSLLRELG PVVAALLFAG RAGSALTAEI GLMRATEQLS SMEMMAVDPL RRVISPRFWA GVISLPLLTV IFVAVGIWGG SLVGVSWKGI DSGFFWSAMQ NAVDWRMDLV NCLIKSVVFA ITVTWISLFN GYDAIPTSAG ISRATTRTVV HSSLAVLGLD FVLTALMFGN

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Macromolecule #2: MlaF

MacromoleculeName: MlaF / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEQSVANLVD MRDVSFTRG N RCIFDNIS LT VPRGKIT AIM GPSGIG KTTL LRLIG GQIAP DHGE ILFDGE NIP AMSRSRL YT VRKRMSML F QSGALFTDM NVFDNVAYPL REHTQLPAP L LHSTVMMK LE AVGLRGA AKL MPSELS GGMA RRAAL ...String:
MEQSVANLVD MRDVSFTRG N RCIFDNIS LT VPRGKIT AIM GPSGIG KTTL LRLIG GQIAP DHGE ILFDGE NIP AMSRSRL YT VRKRMSML F QSGALFTDM NVFDNVAYPL REHTQLPAP L LHSTVMMK LE AVGLRGA AKL MPSELS GGMA RRAAL ARAIA LEPD LIMFDE PFV GQDPITM GV LVKLISEL N SALGVTCVV VSHDVPEVLS IADHAWILA D KKIVAHGS AQ ALQANPD PRV RQFLDG IADG PVPFR YPAGD YHAD LLPGS

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Macromolecule #3: MlaD

MacromoleculeName: MlaD / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHQHQ HENLYFQGMQ TKKNEIWV GIFLLAALLA ALFVCLKAA N VTSIRTEP TY TLYATFD NIG GLKARS PVSI GGVVV GRVAD ITLD PKTYLP RVT LEIEQRY NH IPDTSSLS I RTSGLLGEQ YLALNVGFED PELGTAILK D GDTIQDTK SA ...String:
MHHHHHHQHQ HENLYFQGMQ TKKNEIWV GIFLLAALLA ALFVCLKAA N VTSIRTEP TY TLYATFD NIG GLKARS PVSI GGVVV GRVAD ITLD PKTYLP RVT LEIEQRY NH IPDTSSLS I RTSGLLGEQ YLALNVGFED PELGTAILK D GDTIQDTK SA MVLEDLI GQF LYGSKG DDNK NSGDA PAAAP GNNE TTEPVG TTK

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Macromolecule #4: MlaB

MacromoleculeName: MlaB / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSESLSWMQT GDTLALSGE L DQDVLLPL WE MREEAVK GIT CIDLSR VSRV DTGGL ALLLH LIDL AKKQGN NVT LQGVNDK VY TLAKLYNL P ADVLPR

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Macromolecule #5: Foldon-MlaC

MacromoleculeName: Foldon-MlaC / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHPGS GYIPEAPRDG QAYVRKDGEW VLLSTFLSSG GGSGGGSGGG SSSGGGSGGG SGGGSSSGGG SGGGSGGGSA DQTNPYKLMD EAAQKTFDRL KNEQPQIRAN PDYLRTIVDQ ELLPYVQVKY AGALVLGQYY KSATPAQREA YFAAFREYLK QAYGQALAMY ...String:
MHHHHHHPGS GYIPEAPRDG QAYVRKDGEW VLLSTFLSSG GGSGGGSGGG SSSGGGSGGG SGGGSSSGGG SGGGSGGGSA DQTNPYKLMD EAAQKTFDRL KNEQPQIRAN PDYLRTIVDQ ELLPYVQVKY AGALVLGQYY KSATPAQREA YFAAFREYLK QAYGQALAMY HGQTYQIAPE QPLGDKTIVP IRVTIIDPNG RPPVRLDFQW RKNSQTGNWQ AYDMIAEGVS MITTKQNEWG TLLRTKGIDG LTAQLKSISQ QKITLEEKK

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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

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMNa2HPO4-7H2Osodium phosphate
137.0 mMNaClSodium chloridesodium chloride
2.7 mMKClpotassium chloride
1.5 mMKH2PO4potassium phosphate
0.25 mMC24H46O11n-dodecyl-beta-D-maltoside

Details: 10 mM Na2HPO4.7H2O, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 0.25 mM DDM
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2 / Details: Pixel size = 0.5480 Angstrom
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3769521
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 9 / Software - Name: cryoSPARC (ver. 3.2)
Details: Classes 1, 2 and 4 were combined and refined (Map 1) Classes 3 and 7 were combined and refined (Map 2) Classes 4 was refined (Map 3) Classes 5 was refined (Map 4) Details for Map 1 are ...Details: Classes 1, 2 and 4 were combined and refined (Map 1) Classes 3 and 7 were combined and refined (Map 2) Classes 4 was refined (Map 3) Classes 5 was refined (Map 4) Details for Map 1 are provided below, and Maps 2, 3 and 4 are provided as additional maps
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Details: Map 1 / Number images used: 228579

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

56fx


Chain - Source name: ModelArchive / Chain - Initial model type: in silico model / Details: computed with AlphaFold2

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