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- PDB-9n8x: Intermembrane lipid transport complex LetAB from Escherichia coli... -

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Basic information

Entry
Database: PDB / ID: 9n8x
TitleIntermembrane lipid transport complex LetAB from Escherichia coli (Composite model corresponding to Map 2)
Components
  • Intermembrane transport protein YebS
  • Intermembrane transport protein YebT
KeywordsLIPID TRANSPORT / Lipid transporter / Outer membrane integrity / MCE system / Metal-binding protein / Intermembrane complex
Function / homology
Function and homology information


intermembrane lipid transfer / membrane organization / intracellular transport / outer membrane-bounded periplasmic space / response to heat / identical protein binding / plasma membrane
Similarity search - Function
Intermembrane transport protein PqiA-like, Enterobacteria-type / Intermembrane transport protein PqiA-like / Paraquat-inducible protein A / : / Mce/MlaD / MlaD protein / Multiheme cytochrome c family profile.
Similarity search - Domain/homology
DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / Lipophilic envelope-spanning tunnel protein A / Lipophilic envelope-spanning tunnel protein B
Similarity search - Component
Biological speciesEscherichia coli str. K-12 substr. MG1655 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsSantarossa, C.C. / Bhabha, G. / Ekiert, D.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1K99GM157496-01 United States
Other privateThe Charles H. Revson Foundation
CitationJournal: bioRxiv / Year: 2025
Title: LetA defines a structurally distinct transporter family involved in lipid trafficking.
Authors: Cristina C Santarossa / Yupeng Li / Sara Yousef / Hale S Hasdemir / Carlos C Rodriguez / Max B Haase / Minkyung Baek / Nicolas Coudray / John G Pavek / Kimber N Focke / Annika L Silverberg / ...Authors: Cristina C Santarossa / Yupeng Li / Sara Yousef / Hale S Hasdemir / Carlos C Rodriguez / Max B Haase / Minkyung Baek / Nicolas Coudray / John G Pavek / Kimber N Focke / Annika L Silverberg / Carmelita Bautista / Johannes Yeh / Michael T Marty / David Baker / Emad Tajkhorshid / Damian C Ekiert / Gira Bhabha
Abstract: Membrane transport proteins translocate diverse cargos, ranging from small sugars to entire proteins, across cellular membranes. A few structurally distinct protein families have been described that ...Membrane transport proteins translocate diverse cargos, ranging from small sugars to entire proteins, across cellular membranes. A few structurally distinct protein families have been described that account for most of the known membrane transport processes. However, many membrane proteins with predicted transporter functions remain uncharacterized. We determined the structure of LetAB, a phospholipid transporter involved in outer membrane integrity, and found that LetA adopts a distinct architecture that is structurally and evolutionarily unrelated to known transporter families. LetA functions as a pump at one end of a ~225 Å long tunnel formed by its binding partner, MCE protein LetB, creating a pathway for lipid transport between the inner and outer membranes. Unexpectedly, the LetA transmembrane domains adopt a fold that is evolutionarily related to the eukaryotic tetraspanin family of membrane proteins, including TARPs and claudins. LetA has no detectable homology to known transport proteins, and defines a new class of membrane transporters. Through a combination of deep mutational scanning, molecular dynamics simulations, AlphaFold-predicted alternative states, and functional studies, we present a model for how the LetA-like family of membrane transporters may use energy from the proton-motive force to drive the transport of lipids across the bacterial cell envelope.
History
DepositionFeb 10, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Intermembrane transport protein YebS
B: Intermembrane transport protein YebT
C: Intermembrane transport protein YebT
D: Intermembrane transport protein YebT
E: Intermembrane transport protein YebT
F: Intermembrane transport protein YebT
G: Intermembrane transport protein YebT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)621,92410
Polymers621,1017
Non-polymers8233
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Intermembrane transport protein YebS / Intermembrane transport protein LetA


Mass: 50667.746 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli str. K-12 substr. MG1655 (bacteria)
Strain: MG1655 / Gene: yebS, b1833, JW1822 / Plasmid: Addgene #175960 / Production host: Escherichia coli (E. coli) / Strain (production host): OverExpress C43(DE3) / References: UniProt: P0AD03
#2: Protein
Intermembrane transport protein YebT / Intermembrane transport protein LetB


Mass: 95072.211 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli str. K-12 substr. MG1655 (bacteria)
Strain: MG1655 / Gene: yebT, b1834, JW1823 / Plasmid: pBAD / Details (production host): Addgene #175960 / Production host: Escherichia coli (E. coli) / Strain (production host): OverExpress C43(DE3) / References: UniProt: P76272
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PEF / DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / 3-[AMINOETHYLPHOSPHORYL]-[1,2-DI-PALMITOYL]-SN-GLYCEROL


Mass: 691.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C37H74NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY
Has protein modificationN

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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

ComponentName: LetAB complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.618 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MG1655
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: OverExpress C43(DE3) / Plasmid: Addgene #175960
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane hydrochloride1
2150 mMsodium chlorideNaCl1
30.5 mMn-Dodecyl-B-D-maltoside1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 1300 nm / Calibrated defocus max: 3600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12455

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2PHENIX1.21.2_5419:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6974846
Details: Particles were selected by template-based auto-picking in RELION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 243255 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Model was fit as a rigid-body into the individual map using Chimera. Real space refinement was carried out in PHENIX. Models were then manually inspected and adjusted in Coot. Iterative ...Details: Model was fit as a rigid-body into the individual map using Chimera. Real space refinement was carried out in PHENIX. Models were then manually inspected and adjusted in Coot. Iterative rounds of model building and refinement were performed.
Atomic model buildingPDB-ID: 9N8W

9n8w


Accession code: 9N8W
Details: The initial model consisted of the complete biological assembly of PDB entry 9N8W
Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01242337
ELECTRON MICROSCOPYf_angle_d0.65357477
ELECTRON MICROSCOPYf_dihedral_angle_d9.25815587
ELECTRON MICROSCOPYf_chiral_restr0.0586652
ELECTRON MICROSCOPYf_plane_restr0.0057425

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