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- EMDB-25066: Structure of E. coli LetB delta (Ring6) mutant, Ring1 in the clos... -

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Basic information

Entry
Database: EMDB / ID: EMD-25066
TitleStructure of E. coli LetB delta (Ring6) mutant, Ring1 in the closed state (Model 1)
Map dataFull delta(Ring6) LetB map
Sample
  • Complex: Homohexameric complex of delta(Ring6) mutant form of LetB
    • Protein or peptide: MCE family protein, Intermembrane transport protein YebT chimera
  • Ligand: UNKNOWN ATOM OR ION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsVieni C / Coudray N / Bhabha G / Ekiert D
Funding support United States, 4 items
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, 2021-
Header (metadata) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7see
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25066.png

Downloads & links

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Map

FileDownload / File: emd_25066.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull delta(Ring6) LetB map
Voxel sizeX=Y=Z: 1.272 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.008540669 - 0.067259036
Average (Standard dev.)0.00023162246 (±0.0017352103)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2721.2721.272
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z508.800508.800508.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0090.0670.000

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Supplemental data

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Mask #1

Fileemd_25066_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post processed map from RELION

Fileemd_25066_additional_1.map
AnnotationPost processed map from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map #1

Fileemd_25066_half_map_1.map
AnnotationHalf Map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map #2

Fileemd_25066_half_map_2.map
AnnotationHalf Map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homohexameric complex of delta(Ring6) mutant form of LetB

EntireName: Homohexameric complex of delta(Ring6) mutant form of LetB
Components
  • Complex: Homohexameric complex of delta(Ring6) mutant form of LetB
    • Protein or peptide: MCE family protein, Intermembrane transport protein YebT chimera
  • Ligand: UNKNOWN ATOM OR ION

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Supramolecule #1: Homohexameric complex of delta(Ring6) mutant form of LetB

SupramoleculeName: Homohexameric complex of delta(Ring6) mutant form of LetB
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MG1655
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2(DE3) / Recombinant plasmid: pBEL2004
Molecular weightTheoretical: 473.25 KDa

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

MacromoleculeName: MCE family protein, Intermembrane transport protein YebT chimera
type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 78.967773 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQDRGNTVTI DFMSADGIVP GRTPVRYQGV EVGTVQDISL SDDLRKIEVK VSIKSDMKDA LREETQFWLV TPKASLAGVS GLDALVGGN YIGMMPGKGK EQDHFVALDT QPKYRLDNGD LMIHLQAPDL GSLNSGSLVY FRKIPVGKVY DYAINPNKQG V VIDVLIER ...String:
MQDRGNTVTI DFMSADGIVP GRTPVRYQGV EVGTVQDISL SDDLRKIEVK VSIKSDMKDA LREETQFWLV TPKASLAGVS GLDALVGGN YIGMMPGKGK EQDHFVALDT QPKYRLDNGD LMIHLQAPDL GSLNSGSLVY FRKIPVGKVY DYAINPNKQG V VIDVLIER RFTDLVKKGS RFWNVSGVDA NVSISGAKVK LESLAALVNG AIAFDSPEES KPAEAEDTFG LYEDLAHSQR GV IIKLELP SGAGLTADST PLMYQGLEVG QLTKLDLNPG GKVTGEMTVD PSVVTLLREN TRIELRNPKL SLSDANLSAL LTG KTFELV PGDGEPRKEF VVVPGEKALL HEPDVLTLTL TAPESYGIDA GQPLILHGVQ VGQVIDRKLT SKGVTFTVAI EPQH RELVK GDSKFVVNSR VDVKVGLDGV EFLGASASEW INGGIRILPG DKGEMKASYP LYANLEKALE NSLSDLPTTT VSLSA ETLP DVQAGSVVLY RKFEVGEVIT VRPRANAFDI DLHIKPEYRN LLTSNSVFWA EGGAKVQLNG SGLTVQASPL SRALKG AIS FDNLSGASAS QRKGDKRILY ASETAARAVG LSIIVEAPEA GSLGIGTPVL FRGLEVGTVT GMTLGTLSDR VMIAMRI SK RYQHLVRNNS VFWLASGYSL DFGLTGGVVK TGTFNQFIRG GIAFATPPGT PLAPKAQEGK HFLLQESEPK EWREWGTA L PKGETHHHHH H

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Macromolecule #2: UNKNOWN ATOM OR ION

MacromoleculeName: UNKNOWN ATOM OR ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: UNX

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
20.0 mMC4H11NO3Tris

Details: This buffer was used as gel filtration buffer
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 second blot time and blot force of 5 before plunge freezing.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 10763 / Average exposure time: 1.5 sec. / Average electron dose: 56.3 e/Å2
Details: Images were collected in super resolution mode with a pixel size of 0.318A
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 739130
Details: Template picking followed by iterative rounds of 2D classification
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6v0d


Details: WT LetB
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 152227 / Software - Name: RELION (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 118329
DetailsImages were motion corrected in RELION 3.0
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6v0d

chain_id: A, residue_range: 46-514

6v0d

chain_id: A, residue_range: 747-877
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7see:
Structure of E. coli LetB delta (Ring6) mutant, Ring1 in the closed state (Model 1)

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