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- PDB-9n8w: Intermembrane lipid transport complex LetAB from Escherichia coli... -

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Basic information

Entry
Database: PDB / ID: 9n8w
TitleIntermembrane lipid transport complex LetAB from Escherichia coli (Crosslinked, Composite model corresponding to Map 1)
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
Lipophilic envelope-spanning tunnel protein A / Lipophilic envelope-spanning tunnel protein B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
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 1.0Jul 16, 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)621,2329
Polymers621,1017
Non-polymers1312
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 (E. coli) / Strain: str. K-12 substr. MG1655 / Gene: yebS, b1833, JW1822 / Plasmid: Addgene #175960 / Production host: Escherichia coli B (bacteria) / 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 (E. coli) / Strain: str. K-12 substr. MG1655 / Gene: yebT, b1834, JW1823 / Plasmid: 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
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 crosslinked with glutaraldehyde / 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 hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
30.5 mMn-Dodecyl-B-D-maltosideDDM1
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: 2100 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 100 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12464

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3582925
Details: Particles were selected by template-based auto-picking in cryoSPARC
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158666 / 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 building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
16V0J

6v0j

6V0JThe initial model consisted of the complete biological assembly for PDB entry 6V0J1PDBexperimental model
26V0F

6v0f

6V0FThe initial model consisted of the complete biological assembly for PDB entry 6V0F2PDBexperimental model
36V0E

6v0e

6V0EThe initial model consisted of the complete biological assembly for PDB entry 6V0E3PDBexperimental model
4LetAThe initial model consisted of the complete biological assembly for RoseTTAFold LetA4RoseTTAFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00342592
ELECTRON MICROSCOPYf_angle_d0.51657825
ELECTRON MICROSCOPYf_dihedral_angle_d8.12215693
ELECTRON MICROSCOPYf_chiral_restr0.0446682
ELECTRON MICROSCOPYf_plane_restr0.0057484

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