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- PDB-6vci: Lipophilic envelope-spanning tunnel protein (LetB), domains MCE2-MCE3 -

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Entry
Database: PDB / ID: 6vci
TitleLipophilic envelope-spanning tunnel protein (LetB), domains MCE2-MCE3
ComponentsLipophilic envelope-spanning tunnel protein LetB
KeywordsLIPID TRANSPORT / bacterial cell envelope / MCE / YebT / LetB
Function / homologyMce/MlaD / MlaD protein / intermembrane lipid transfer / outer membrane-bounded periplasmic space / identical protein binding / plasma membrane / Intermembrane transport protein YebT
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsIsom, G.L. / Coudray, N. / MacRae, M.R. / McManus, C.T. / Ekiert, D.C. / Bhabha, G.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112982 United States
Other privateDFS-20-16 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777-01 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ACB-12002 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)AGM-12006 United States
CitationJournal: Cell / Year: 2020
Title: LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope.
Authors: Georgia L Isom / Nicolas Coudray / Mark R MacRae / Collin T McManus / Damian C Ekiert / Gira Bhabha /
Abstract: Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate ...Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.
History
DepositionDec 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 6, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
C: Lipophilic envelope-spanning tunnel protein LetB
A: Lipophilic envelope-spanning tunnel protein LetB


Theoretical massNumber of molelcules
Total (without water)50,8402
Polymers50,8402
Non-polymers00
Water86548
1
C: Lipophilic envelope-spanning tunnel protein LetB


Theoretical massNumber of molelcules
Total (without water)25,4201
Polymers25,4201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Lipophilic envelope-spanning tunnel protein LetB


Theoretical massNumber of molelcules
Total (without water)25,4201
Polymers25,4201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)87.560, 87.560, 116.990
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65
Space group name HallP65
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: -x,-y,z+1/2

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Components

#1: Protein Lipophilic envelope-spanning tunnel protein LetB / Intermembrane transport protein YebT


Mass: 25419.844 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: yebT, b1834, JW1823 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P76272
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 48 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.68 Å3/Da / Density % sol: 54.07 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Crystals grew from drops consisting of 100 nL protein plus 100 nL of a reservoir solution consisting of 0.1 M Tris pH 8.5, 20% w/v PEG 1K, and were cryoprotected by supplementing the ...Details: Crystals grew from drops consisting of 100 nL protein plus 100 nL of a reservoir solution consisting of 0.1 M Tris pH 8.5, 20% w/v PEG 1K, and were cryoprotected by supplementing the reservoir solution with 15% ethylene glycol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.0332 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 29, 2019
RadiationMonochromator: double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 2.15→46.32 Å / Num. obs: 27654 / % possible obs: 99.8 % / Redundancy: 9.4 % / CC1/2: 1 / Rrim(I) all: 0.05 / Net I/σ(I): 21.3
Reflection shellResolution: 2.15→2.21 Å / Num. unique obs: 26145 / CC1/2: 0.62 / Rrim(I) all: 1.26 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6V0F

6v0f


Resolution: 2.15→41 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflectionSelection details
Rfree0.2436 1321 5.05 %Random
Rwork0.2136 ---
obs-26145 99.8 %-
Refinement stepCycle: LAST / Resolution: 2.15→41 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3246 0 0 48 3294
LS refinement shellResolution: 2.1502→2.2362 Å
RfactorNum. reflection% reflection
Rfree0.3755 --
Rwork0.3477 --
obs-2429 79 %

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