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- EMDB-35253: Cryo-EM Structure of OmpC3-MlaA-MlaC Complex in MSP2N2 Nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-35253
TitleCryo-EM Structure of OmpC3-MlaA-MlaC Complex in MSP2N2 Nanodiscs
Map data
Sample
  • Complex: OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC
    • Complex: Outer membrane porin C
      • Protein or peptide: Outer membrane porin C
    • Complex: Complex of MlaA disulfide trapped with MlaC
      • Protein or peptide: Intermembrane phospholipid transport system lipoprotein MlaA
      • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaC
  • Ligand: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{R})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-5-oxidanyl-oxan-4-yl]oxy-4,5-bis(oxidanyl)oxane-2-carboxylic acid
Keywordsbacteria / outer membrane / phospholipid / lipid asymmetry / membrane protein / protein complex structure / channel / LIPID TRANSPORT
Function / homology
Function and homology information


intermembrane phospholipid transfer / phospholipid transport / porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane / virus receptor activity / outer membrane-bounded periplasmic space / receptor-mediated virion attachment to host cell / DNA damage response ...intermembrane phospholipid transfer / phospholipid transport / porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane / virus receptor activity / outer membrane-bounded periplasmic space / receptor-mediated virion attachment to host cell / DNA damage response / identical protein binding / metal ion binding
Similarity search - Function
MlaA lipoprotein / MlaA lipoprotein / Tgt2/MlaC superfamily / Toluene tolerance Ttg2/phospholipid-binding protein MlaC / MlaC protein / Porin, gammaproteobacterial / Porin, Gram-negative type, conserved site / General diffusion Gram-negative porins signature. / Gram-negative porin / Porin, Gram-negative type ...MlaA lipoprotein / MlaA lipoprotein / Tgt2/MlaC superfamily / Toluene tolerance Ttg2/phospholipid-binding protein MlaC / MlaC protein / Porin, gammaproteobacterial / Porin, Gram-negative type, conserved site / General diffusion Gram-negative porins signature. / Gram-negative porin / Porin, Gram-negative type / Porin domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Outer membrane porin C / Intermembrane phospholipid transport system binding protein MlaC / Intermembrane phospholipid transport system lipoprotein MlaA
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsYeow J / Luo M / Chng SS
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Other governmentMOH-000145 Singapore
CitationJournal: Nat Commun / Year: 2023
Title: Molecular mechanism of phospholipid transport at the bacterial outer membrane interface.
Authors: Jiang Yeow / Min Luo / Shu-Sin Chng /
Abstract: The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer with outer leaflet lipopolysaccharides and inner leaflet phospholipids (PLs). This unique lipid asymmetry renders the ...The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer with outer leaflet lipopolysaccharides and inner leaflet phospholipids (PLs). This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. To maintain this barrier, the OmpC-Mla system removes mislocalized PLs from the OM outer leaflet, and transports them to the inner membrane (IM); in the first step, the OmpC-MlaA complex transfers PLs to the periplasmic chaperone MlaC, but mechanistic details are lacking. Here, we biochemically and structurally characterize the MlaA-MlaC transient complex. We map the interaction surfaces between MlaA and MlaC in Escherichia coli, and show that electrostatic interactions are important for MlaC recruitment to the OM. We further demonstrate that interactions with MlaC modulate conformational states in MlaA. Finally, we solve a 2.9-Å cryo-EM structure of a disulfide-trapped OmpC-MlaA-MlaC complex in nanodiscs, reinforcing the mechanism of MlaC recruitment, and highlighting membrane thinning as a plausible strategy for directing lipids for transport. Our work offers critical insights into retrograde PL transport by the OmpC-Mla system in maintaining OM lipid asymmetry.
History
DepositionFeb 5, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35253.png

Downloads & links

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Map

FileDownload / File: emd_35253.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.18563645 - 0.45763534
Average (Standard dev.)0.0039384207 (±0.017781612)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35253_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_35253_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_35253_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC

EntireName: OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC
Components
  • Complex: OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC
    • Complex: Outer membrane porin C
      • Protein or peptide: Outer membrane porin C
    • Complex: Complex of MlaA disulfide trapped with MlaC
      • Protein or peptide: Intermembrane phospholipid transport system lipoprotein MlaA
      • Protein or peptide: Intermembrane phospholipid transport system binding protein MlaC
  • Ligand: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{R})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-5-oxidanyl-oxan-4-yl]oxy-4,5-bis(oxidanyl)oxane-2-carboxylic acid

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Supramolecule #1: OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC

SupramoleculeName: OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Outer membrane complex of 3OmpC with disulfide-trapped MlaA and MlaC reconstituted in Escherichia coli polar lipids and MSP2N2 nanodiscs
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / Location in cell: Outer membrane
Molecular weightTheoretical: 52 KDa

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Supramolecule #2: Outer membrane porin C

SupramoleculeName: Outer membrane porin C / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Trimeric OmpC porins in complex with MlaA at the outer membrane disulfide-trapped with MlaC
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / Location in cell: Outer membrane

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Supramolecule #3: Complex of MlaA disulfide trapped with MlaC

SupramoleculeName: Complex of MlaA disulfide trapped with MlaC / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Intermembrane phospholipid transport system lipoprotein MlaA disulfide trapped with MlaC
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / Location in cell: Outer membrane

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Macromolecule #1: Outer membrane porin C

MacromoleculeName: Outer membrane porin C / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 38.336242 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: AEVYNKDGNK LDLYGKVDGL HYFSDNKDVD GDQTYMRLGF KGETQVTDQL TGYGQWEYQI QGNSAENENN SWTRVAFAGL KFQDVGSFD YGRNYGVVYD VTSWTDVLPE FGGDTYGSDN FMQQRGNGFA TYRNTDFFGL VDGLNFAVQY QGKNGNPSGE G FTSGVTNN ...String:
AEVYNKDGNK LDLYGKVDGL HYFSDNKDVD GDQTYMRLGF KGETQVTDQL TGYGQWEYQI QGNSAENENN SWTRVAFAGL KFQDVGSFD YGRNYGVVYD VTSWTDVLPE FGGDTYGSDN FMQQRGNGFA TYRNTDFFGL VDGLNFAVQY QGKNGNPSGE G FTSGVTNN GRDALRQNGD GVGGSITYDY EGFGIGGAIS SSKRTDAQNT AAYIGNGDRA ETYTGGLKYD ANNIYLAAQY TQ TYNATRV GSLGWANKAQ NFEAVAQYQF DFGLRPSLAY LQSKGKNLGR GYDDEDILKY VDVGATYYFN KNMSTYVDYK INL LDDNQF TRDAGINTDN IVALGLVYQF

UniProtKB: Outer membrane porin C

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Macromolecule #2: Intermembrane phospholipid transport system lipoprotein MlaA

MacromoleculeName: Intermembrane phospholipid transport system lipoprotein MlaA
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 26.298336 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: CASSGTDQQG RSDPLEGFNR TMYNFNFNVL DPYIVRPVAV AWRDYVPQPA RNGLSNFTGN LEEPAVMVNY FLQGDPYQGM VHFTRFFLN TILGMGGFID VAGMANPKLQ RTEPHRFGST LGHYGVGYGP YVQLPFYGSF TLRDDGGDMA DGFYPVLSWL T WPMSVGKW ...String:
CASSGTDQQG RSDPLEGFNR TMYNFNFNVL DPYIVRPVAV AWRDYVPQPA RNGLSNFTGN LEEPAVMVNY FLQGDPYQGM VHFTRFFLN TILGMGGFID VAGMANPKLQ RTEPHRFGST LGHYGVGYGP YVQLPFYGSF TLRDDGGDMA DGFYPVLSWL T WPMSVGKW TLEGIETRAQ LLDSDGLLRC SSDPYIMVRE AYFQRHDFIA NGGELKPQEN PNAQAIQDDL KDIDSE

UniProtKB: Intermembrane phospholipid transport system lipoprotein MlaA

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Macromolecule #3: Intermembrane phospholipid transport system binding protein MlaC

MacromoleculeName: Intermembrane phospholipid transport system binding protein MlaC
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 22.903922 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: AADQTNPYKL MDEAAQKTFD RLKNEQPQIR ANPDYLRTIV DQELLPYVQV KYAGALVLGQ YYKSATPAQR EAYFAAFREY LKQAYGQAL AMYHGQTYQI APEQPLGDKT IVPIRVTIID PNGRPPVRLD FQWRKNSQTG NWQAYDMIAE GCSMITTKQN E WGTLLRTK ...String:
AADQTNPYKL MDEAAQKTFD RLKNEQPQIR ANPDYLRTIV DQELLPYVQV KYAGALVLGQ YYKSATPAQR EAYFAAFREY LKQAYGQAL AMYHGQTYQI APEQPLGDKT IVPIRVTIID PNGRPPVRLD FQWRKNSQTG NWQAYDMIAE GCSMITTKQN E WGTLLRTK GIDGLTAQLK SISQQKITLE EKKLEHHHHH H

UniProtKB: Intermembrane phospholipid transport system binding protein MlaC

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Macromolecule #4: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(...

MacromoleculeName: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3- ...Name: (2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{R})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-5-oxidanyl-oxan-4-yl]oxy-4,5-bis(oxidanyl)oxane-2-carboxylic acid
type: ligand / ID: 4 / Number of copies: 3 / Formula: KDL
Molecular weightTheoretical: 2.238718 KDa
Chemical component information

ChemComp-KDL:
(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[(2~{R},4~{R},5~{R},6~{R})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{R})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-5-oxidanyl-oxan-4-yl]oxy-4,5-bis(oxidanyl)oxane-2-carboxylic acid

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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

Concentration12 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloridesodium chloride

Details: Tris-buffered saline (TBS) buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl)
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Tridiem 4K / Energy filter - Slit width: 20 eV
Details: Gatan GIF post-column energy filter operated in zero-loss mode
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 6018 / Average exposure time: 5.99 sec. / Average electron dose: 90.0 e/Å2
Details: Images were collected in movie-mode at 50 frames per image
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2434319
Details: We performed automated particle picking using Blob and Template Picker for initial template picking and final sampling respectively.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.0)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.0.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.0) / Number images used: 76463
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

5nup

chain_id: A, residue_range: 1-367, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA

5nup

chain_id: B, residue_range: 1-367, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA

5nup

chain_id: C, residue_range: 1-367, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA

5nup

chain_id: D, residue_range: 1-251, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA

5uwa

chain_id: A, residue_range: 1-211, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 69.1 / Target criteria: Cross-correlation coefficient
Output model

PDB-8i8x:
Cryo-EM Structure of OmpC3-MlaA-MlaC Complex in MSP2N2 Nanodiscs

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