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

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Basic information

Entry
Database: PDB / ID: 8i8x
TitleCryo-EM Structure of OmpC3-MlaA-MlaC Complex in MSP2N2 Nanodiscs
Components
  • Intermembrane phospholipid transport system binding protein MlaC
  • Intermembrane phospholipid transport system lipoprotein MlaA
  • Outer membrane porin C
KeywordsLIPID TRANSPORT / bacteria / outer membrane / phospholipid / lipid asymmetry / membrane protein / protein complex structure / channel
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
Chem-KDL / Outer membrane porin C / Intermembrane phospholipid transport system binding protein MlaC / Intermembrane phospholipid transport system lipoprotein MlaA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsYeow, J. / Luo, M. / Chng, S.S.
Funding support Singapore, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer membrane porin C
B: Outer membrane porin C
C: Outer membrane porin C
D: Intermembrane phospholipid transport system lipoprotein MlaA
F: Intermembrane phospholipid transport system binding protein MlaC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,9278
Polymers164,2115
Non-polymers6,7163
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, In vivo site-specific para-L-Benzoyl-phenylalanine UV-dependent, and disulfide photocrosslinking to determine MlaA and MlaC interaction, light scattering, Size-exclusion ...Evidence: cross-linking, In vivo site-specific para-L-Benzoyl-phenylalanine UV-dependent, and disulfide photocrosslinking to determine MlaA and MlaC interaction, light scattering, Size-exclusion chromatography/Multi-angle light scattering to determine the size of OmpC3-MlaA-MlaC complex in DDM, native gel electrophoresis, Native-PAGE and 2D-PAGE to confirm the presence of the OmpC3-MlaA-MlaC complex in MSP2N2 nanodiscs
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer membrane porin C / Outer membrane protein 1B / Outer membrane protein C / Porin OmpC


Mass: 38336.242 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: ompC, meoA, par, b2215, JW2203 / Plasmid: pDSW206 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): LAMBDADE3 / References: UniProt: P06996
#2: Protein Intermembrane phospholipid transport system lipoprotein MlaA


Mass: 26298.336 Da / Num. of mol.: 1 / Mutation: Q205C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: mlaA, vacJ, b2346, JW2343 / Plasmid: pCloDF / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): LAMBDADE3 / References: UniProt: P76506
#3: Protein Intermembrane phospholipid transport system binding protein MlaC


Mass: 22903.922 Da / Num. of mol.: 1 / Mutation: V171C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: mlaC, yrbC, b3192, JW3159 / Plasmid: pCloDF / Details (production host): MCS1 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): LAMDADE3 / References: UniProt: P0ADV7
#4: Chemical 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


Mass: 2238.718 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C110H202N2O39P2
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1OmpC3-MlaA complex in MSP2N2 nanodiscs disulfide-bonded to MlaCCOMPLEXOuter membrane complex of 3OmpC with disulfide-trapped MlaA and MlaC reconstituted in Escherichia coli polar lipids and MSP2N2 nanodiscs#1-#30RECOMBINANT
2Outer membrane porin CCOMPLEXTrimeric OmpC porins in complex with MlaA at the outer membrane disulfide-trapped with MlaC#11RECOMBINANT
3Complex of MlaA disulfide trapped with MlaCCOMPLEXIntermembrane phospholipid transport system lipoprotein MlaA disulfide trapped with MlaC#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.251 MDaNO
210.12 MDaNO
310.052 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellular location
21Escherichia coli (E. coli)83333K12Outer membrane
32Escherichia coli (E. coli)83333K12Outer membrane
43Escherichia coli (E. coli)83333K12Outer membrane
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)511693BL21pDSW206
32Escherichia coli (E. coli)511693BL21pCloDF
43Escherichia coli (E. coli)511693BL21pCloDF
Buffer solutionpH: 8
Details: Tris-buffered saline (TBS) buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl)
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.99 sec. / Electron dose: 90 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6018
Details: Images were collected in movie-mode at 50 frames per image
EM imaging opticsEnergyfilter name: GIF Tridiem 4K
Details: Gatan GIF post-column energy filter operated in zero-loss mode
Energyfilter slit width: 20 eV
Image scansSampling size: 5.2 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.0particle selection
2SerialEMimage acquisition
4cryoSPARC4.0.0CTF correction
7UCSF Chimera1.15model fitting
9cryoSPARC4.0.0initial Euler assignment
10cryoSPARC4.0.0final Euler assignment
11cryoSPARC4.0.0classification
12cryoSPARC4.0.03D reconstruction
13Coot0.9.4.1 ELmodel refinement
14PHENIX1.20.1-4487model refinement
CTF correctionDetails: Patch CTF routine in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2434319
Details: We performed automated particle picking using Blob and Template Picker for initial template picking and final sampling respectively.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76463 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 69.1 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building

3D fitting-ID: 1 / Details: The initial model consisted of the chains A,B,C,D, and chain A for PDB entry 5UWA / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue range
15NUP

5nup

A5NUPA1-36711-367
25NUP

5nup

B5NUPB1-36711-367
35NUP

5nup

C5NUPC1-36711-367
45NUP

5nup

D5NUPD1-25111-251
55UWA

5uwa

A5UWAA1-21121-211
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00711462
ELECTRON MICROSCOPYf_angle_d1.17415543
ELECTRON MICROSCOPYf_dihedral_angle_d17.2421802
ELECTRON MICROSCOPYf_chiral_restr0.0691625
ELECTRON MICROSCOPYf_plane_restr0.0072059

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