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- EMDB-29041: E. coli MlaFEDB bound to MlaC -

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Basic information

Entry
Database: EMDB / ID: EMD-29041
TitleE. coli MlaFEDB bound to MlaC
Map dataMlaFEDB with 2 bound MlaC
Sample
  • Complex: E. coli MlaFEDB bound to MlaC
    • Protein or peptide: MlaE
    • Protein or peptide: MlaF
    • Protein or peptide: MlaD
    • Protein or peptide: MlaB
    • Protein or peptide: MlaC-foldon
KeywordsE. coli / bacteria / outer membrane / cryo-EM / lipid transport / mla / structural biology
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 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)R35GM128777 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
DepositionDec 8, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29041.png

Downloads & links

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Map

FileDownload / File: emd_29041.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMlaFEDB with 2 bound MlaC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 304 pix.
= 333.184 Å		1.1 Å/pix.
x 304 pix.
= 333.184 Å		1.1 Å/pix.
x 304 pix.
= 333.184 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.096 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.34019306 - 0.63718325
Average (Standard dev.)0.0002489991 (±0.03469369)
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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Half map: #2

Fileemd_29041_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_29041_half_map_2.map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli MlaFEDB bound to MlaC

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

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Supramolecule #1: E. coli MlaFEDB bound to MlaC

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

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Macromolecule #2: MlaE

MacromoleculeName: MlaE / type: protein_or_peptide / ID: 2 / 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 #3: MlaF

MacromoleculeName: MlaF / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSML FQSGALFTDM NVFDNVAYPL REHTQLPAPL LHSTVMMKLE AVGLRGAAKL MPSELSGGMA RRAALARAIA LEPDLIMFDE ...String:
MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSML FQSGALFTDM NVFDNVAYPL REHTQLPAPL LHSTVMMKLE AVGLRGAAKL MPSELSGGMA RRAALARAIA LEPDLIMFDE PFVGQDPITM GVLVKLISEL NSALGVTCVV VSHDVPEVLS IADHAWILAD KKIVAHGSAQ ALQANPDPRV RQFLDGIADG PVPFRYPAGD YHADLLPGS

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Macromolecule #4: MlaD

MacromoleculeName: MlaD / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHQHQ HENLYFQGMQ TKKNEIWVGI FLLAALLAAL FVCLKAANVT SIRTEPTYTL YATFDNIGGL KARSPVSIGG VVVGRVADIT LDPKTYLPRV TLEIEQRYNH IPDTSSLSIR TSGLLGEQYL ALNVGFEDPE LGTAILKDGD TIQDTKSAMV LEDLIGQFLY ...String:
MHHHHHHQHQ HENLYFQGMQ TKKNEIWVGI FLLAALLAAL FVCLKAANVT SIRTEPTYTL YATFDNIGGL KARSPVSIGG VVVGRVADIT LDPKTYLPRV TLEIEQRYNH IPDTSSLSIR TSGLLGEQYL ALNVGFEDPE LGTAILKDGD TIQDTKSAMV LEDLIGQFLY GSKGDDNKNS GDAPAAAPGN NETTEPVGTT K

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Macromolecule #5: MlaB

MacromoleculeName: MlaB / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSESLSWMQT GDTLALSGEL DQDVLLPLWE MREEAVKGIT CIDLSRVSRV DTGGLALLLH LIDLAKKQGN NVTLQGVNDK VYTLAKLYNL PADVLPR

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Macromolecule #6: MlaC-foldon

MacromoleculeName: MlaC-foldon / type: protein_or_peptide / ID: 6 / 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 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2 / Details: Pixel size = 0.5480 Angstrom
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3769521
Startup modelType of model: NONE
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 64080
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 9 / Software - Name: cryoSPARC (ver. 3.2)

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