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- PDB-7see: Structure of E. coli LetB delta (Ring6) mutant, Ring1 in the clos... -

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Basic information

Entry
Database: PDB / ID: 7see
TitleStructure of E. coli LetB delta (Ring6) mutant, Ring1 in the closed state (Model 1)
ComponentsMCE family protein, Intermembrane transport protein YebT chimera
KeywordsLIPID TRANSPORT / bacterial cell envelope / MCE
Function / homology
Function and homology information


intermembrane lipid transfer / membrane organization / outer membrane-bounded periplasmic space / identical protein binding / plasma membrane
Similarity search - Function
: / Mce/MlaD / MlaD protein
Similarity search - Domain/homology
: / Lipophilic envelope-spanning tunnel protein B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsVieni, C. / Coudray, N. / Bhabha, G. / Ekiert, D.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777 United States
Damon Runyon Cancer Research FoundationDFS-20-16 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1F30AI154907-01 United States
CitationJournal: J Mol Biol / Year: 2022
Title: Role of Ring6 in the Function of the E. coli MCE Protein LetB.
Authors: Casey Vieni / Nicolas Coudray / Georgia L Isom / Gira Bhabha / Damian C Ekiert /
Abstract: LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In ...LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.
History
DepositionSep 30, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: MCE family protein, Intermembrane transport protein YebT chimera
B: MCE family protein, Intermembrane transport protein YebT chimera
C: MCE family protein, Intermembrane transport protein YebT chimera
D: MCE family protein, Intermembrane transport protein YebT chimera
E: MCE family protein, Intermembrane transport protein YebT chimera
F: MCE family protein, Intermembrane transport protein YebT chimera


Theoretical massNumber of molelcules
Total (without water)473,80712
Polymers473,8076
Non-polymers06
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, electron microscopy, negative stain electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48610 Å2
ΔGint-304 kcal/mol
Surface area200250 Å2

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Components

#1: Protein
MCE family protein, Intermembrane transport protein YebT chimera


Mass: 78967.773 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: HGS75_03440, yebT, b1834, JW1823 / Plasmid: pBEL2004 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2(DE3) / References: UniProt: A0A769F599, UniProt: P76272
#2: Chemical
ChemComp-UNX / UNKNOWN ATOM OR ION


Num. of mol.: 6 / Source method: obtained synthetically
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

ComponentName: Homohexameric complex of delta(Ring6) mutant form of LetB
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.47325 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MG1655
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta 2(DE3) / Plasmid: pBEL2004
Buffer solutionpH: 8 / Details: This buffer was used as gel filtration buffer
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMTrisC4H11NO31
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 96 % / Chamber temperature: 277 K
Details: 3 second blot time and blot force of 5 before plunge freezing

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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: 37000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 56.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10763
Details: Images were collected in super resolution mode with a pixel size of 0.318A
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9model fitting
9PHENIX1.18.2model refinement
10cryoSPARC2.1initial Euler assignment
11RELION3.1.0final Euler assignment
12RELION3.1.0classification
13RELION3.1.03D reconstruction
Image processingDetails: Images were motion corrected in RELION 3.0
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 739130
Details: Template picking followed by iterative rounds of 2D classification
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118329 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 6V0D / Initial refinement model-ID: 1 / Pdb chain-ID: A / PDB-ID: 6V0D

6v0d

/ Source name: PDB / Type: experimental model

IDPdb chain residue range
146-514
2747-877

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