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- PDB-8i8r: Cryo-EM Structure of OmpC3-MlaA Complex in MSP2N2 Nanodiscs -

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Basic information

Entry
Database: PDB / ID: 8i8r
TitleCryo-EM Structure of OmpC3-MlaA Complex in MSP2N2 Nanodiscs
Components
  • 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 / porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane / virus receptor activity / receptor-mediated virion attachment to host cell / DNA damage response / identical protein binding / metal ion binding
Similarity search - Function
MlaA lipoprotein / MlaA lipoprotein / 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 lipoprotein MlaA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,0237
Polymers141,3074
Non-polymers6,7163
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Size exclusion chromatography of OmpC3-MlaA(-MlaC) reconstituted in MSP2N2 nanodiscs, and peak fractions of interest were subjected to heated reducing and non-reducing SDS- ...Evidence: gel filtration, Size exclusion chromatography of OmpC3-MlaA(-MlaC) reconstituted in MSP2N2 nanodiscs, and peak fractions of interest were subjected to heated reducing and non-reducing SDS-PAGE., cross-linking, In-vivo site-specific para-L-Benzoyl-phenylalanine UV-dependent and disulfide crosslinking to determine interaction, light scattering, Size-exclusion chromatography/multi-angle light scattering analysis of OmpC3-MlaA-MlaC-His in DDM
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): LAMDBADE3 / References: UniProt: P76506
#3: 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
1Outer membrane complex of OmpC-MlaA with periplasmic MlaC reconstituted in MSP2N2 nanodiscsCOMPLEXOuter membrane complex of OmpC with disulfide-trapped MlaA and MlaC reconstituted in Escherichia coli polar lipids and MSP2N2 nanodiscs#1-#20RECOMBINANT
2Outer membrane porin CCOMPLEXOmpC#11RECOMBINANT
3Intermembrane phospholipid transport system lipoprotein MlaACOMPLEXMlaA#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.213 MDaNO
210.04 MDaNO
310.028 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)83333K12
32Escherichia coli (E. coli)83333K12
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)511693BL21pDSW206
32Escherichia 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
8Coot0.9.4.1 ELmodel fitting
10cryoSPARC4.0.0initial Euler assignment
11cryoSPARC4.0.0final Euler assignment
12cryoSPARC4.0.0classification
13cryoSPARC4.0.03D reconstruction
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: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205487 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 88 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Building of the high-resolution structures of OmpC-MlaA was done via approximate fitting using the OmpC-MlaA crystal structure (PDB 5NUP) as rigid bodies into the density in Chimera, ...Details: Building of the high-resolution structures of OmpC-MlaA was done via approximate fitting using the OmpC-MlaA crystal structure (PDB 5NUP) as rigid bodies into the density in Chimera, followed by refinement in COOT.
Atomic model building

3D fitting-ID: 1 / Accession code: 5NUP / Details: The initial model consisted of the chains A,B,C,D for PDB entry 5NUP / Initial refinement model-ID: 1 / PDB-ID: 5NUP

5nup

/ Source name: PDB / Type: experimental model

IDPdb chain-IDChain-IDChain residue rangePdb chain residue range
1AA1-3461-346
2BB1-3461-346
3CC1-3461-346
4DD1-2111-211
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610392
ELECTRON MICROSCOPYf_angle_d1.14614055
ELECTRON MICROSCOPYf_dihedral_angle_d17.3521584
ELECTRON MICROSCOPYf_chiral_restr0.0661421
ELECTRON MICROSCOPYf_plane_restr0.0061843

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