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- PDB-7sef: Structure of E. coli LetB delta (Ring6) mutant, Ring 1 in the ope... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7sef | |||||||||||||||
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Title | Structure of E. coli LetB delta (Ring6) mutant, Ring 1 in the open state (Model 2, Rings 1-3 only) | |||||||||||||||
![]() | MCE family protein, Intermembrane transport protein YebT chimera | |||||||||||||||
![]() | LIPID TRANSPORT / bacterial cell envelope / MCE | |||||||||||||||
Function / homology | Mce/MlaD / MlaD protein / intermembrane lipid transfer / outer membrane-bounded periplasmic space / identical protein binding / plasma membrane / : / Intermembrane transport protein YebT![]() | |||||||||||||||
Biological species | ![]() ![]() | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.55 Å | |||||||||||||||
![]() | Vieni, C. / Coudray, N. / Bhabha, G. / Ekiert, D. | |||||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 402.7 KB | Display | ![]() |
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PDB format | ![]() | 317.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.2 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 67.2 KB | Display | |
Data in CIF | ![]() | 90.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 25067MC ![]() 7seeC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 78967.773 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Homohexameric complex of delta (Ring6) mutant form of LetB Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.47325 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||
Buffer solution | pH: 8 / Details: This buffer was used as gel filtration buffer | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse | |||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 37000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 scans | Width: 11520 / Height: 8184 |
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Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Images were motion corrected in RELION 3.0 | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 739130 Details: Template picking followed by iterative rounds of 2D classification | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39547 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6V0D Pdb chain-ID: A / Accession code: 6V0D / Pdb chain residue range: 46-514 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||||||||||||
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