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- EMDB-18464: Cryo-EM structure of MmpL3 from Mycobacterium smegmatis reconstit... -

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Basic information

Entry
Database: EMDB / ID: EMD-18464
TitleCryo-EM structure of MmpL3 from Mycobacterium smegmatis reconstituted into peptidiscs
Map dataPrimary map. Map from non-uniform refinement and density modification (resolution 3.23 Angstrom)
Sample
  • Complex: monomer structure of MmpL3
    • Protein or peptide: Trehalose monomycolate exporter MmpL3
Keywordsmycobacterium / trehalose monomycolate / TMM / peptidisc / MmpL3 / MEMBRANE PROTEIN
Function / homology
Function and homology information


phosphatidylethanolamine transfer activity / phosphatidylglycerol binding / trehalose transmembrane transporter activity / trehalose transport / mycolate cell wall layer assembly / cell wall biogenesis / diacylglycerol binding / cell pole / cell tip / mycolic acid biosynthetic process ...phosphatidylethanolamine transfer activity / phosphatidylglycerol binding / trehalose transmembrane transporter activity / trehalose transport / mycolate cell wall layer assembly / cell wall biogenesis / diacylglycerol binding / cell pole / cell tip / mycolic acid biosynthetic process / cell septum / phospholipid transport / cardiolipin binding / phosphatidylethanolamine binding / phosphatidylinositol binding / regulation of membrane potential / cell wall organization / response to xenobiotic stimulus / response to antibiotic / plasma membrane
Similarity search - Function
: / Membrane transport protein MMPL domain / MMPL family
Similarity search - Domain/homology
Trehalose monomycolate exporter MmpL3
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsCouston J / Guo Z / Wang K / Gourdon PE / Blaise M
Funding support France, Denmark, 3 items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS)CNRS-UCPH joint program France
LundbeckfondenR313-2019-774 Denmark
Agence Nationale de la Recherche (ANR)ANR-17-CE11-0008-01 France
CitationJournal: Curr Res Struct Biol / Year: 2023
Title: Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs.
Authors: Julie Couston / Zongxin Guo / Kaituo Wang / Pontus Gourdon / Mickaël Blaise /
Abstract: Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses ...Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses long acyl chains with up to 90 carbon atoms. TMM represents an essential component of mycobacteria and is synthesized in the cytoplasm, and then flipped over the plasma membrane by a specific transporter known as MmpL3. Over the last decade, MmpL3 has emerged as an attractive drug target to combat mycobacterial infections. Recent three-dimensional structures of MmpL3 determined by X-ray crystallography and cryo-EM have increased our understanding of the TMM transport, and the mode of action of inhibiting compounds. These structures were obtained in the presence of detergent and/or in a lipidic environment. In this study, we demonstrate the possibility of obtaining a high-quality cryo-EM structure of MmpL3 without any presence of detergent through the reconstitution of the protein into peptidiscs. The structure was determined at an overall resolution of 3.2 Å and demonstrates that the overall structure of MmpL3 is preserved as compared to previous structures. Further, the study identified a new structural arrangement of the linker that fuses the two subdomains of the transmembrane domain, suggesting the feature may serve a role in the transport process.
History
DepositionSep 15, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18464.png

Downloads & links

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Map

FileDownload / File: emd_18464.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map. Map from non-uniform refinement and density modification (resolution 3.23 Angstrom)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 320 pix.
= 232. Å		0.73 Å/pix.
x 320 pix.
= 232. Å		0.73 Å/pix.
x 320 pix.
= 232. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.34
Minimum - Maximum-2.8968923 - 4.3449755
Average (Standard dev.)0.00023006967 (±0.07212121)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 232.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18464_msk_1.map
Projections & Slices
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Additional map: map from non-uniform refinement (resolution 3.4 Angstrom)

Fileemd_18464_additional_1.map
Annotationmap from non-uniform refinement (resolution 3.4 Angstrom)
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_18464_half_map_1.map
Projections & Slices
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Histogram

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Half map: #2

Fileemd_18464_half_map_2.map
Projections & Slices
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Sample components

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Entire : monomer structure of MmpL3

EntireName: monomer structure of MmpL3
Components
  • Complex: monomer structure of MmpL3
    • Protein or peptide: Trehalose monomycolate exporter MmpL3

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Supramolecule #1: monomer structure of MmpL3

SupramoleculeName: monomer structure of MmpL3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Macromolecule #1: Trehalose monomycolate exporter MmpL3

MacromoleculeName: Trehalose monomycolate exporter MmpL3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 85.465344 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGFAWWGRTV YQFRYIVIGV MVALCLGGGV YGISLGNHVT QSGFYDEGSQ SVAASLIGDE VYGRDRTSHV VAILTPPDDK KVTDKAWQK KVTEELDQVV KDHEDQIVGW VGWLKAPDTT DPTVSAMKTQ DLRHTFISIP LQGDDDDEIL KNYQVVEPEL Q QVNGGDIR ...String:
MGFAWWGRTV YQFRYIVIGV MVALCLGGGV YGISLGNHVT QSGFYDEGSQ SVAASLIGDE VYGRDRTSHV VAILTPPDDK KVTDKAWQK KVTEELDQVV KDHEDQIVGW VGWLKAPDTT DPTVSAMKTQ DLRHTFISIP LQGDDDDEIL KNYQVVEPEL Q QVNGGDIR LAGLNPLASE LTGTIGEDQK RAEVAAIPLV AVVLFFVFGT VIAAALPAII GGLAIAGALG IMRLVAEFTP VH FFAQPVV TLIGLGIAID YGLFIVSRFR EEIAEGYDTE AAVRRTVMTS GRTVVFSAVI IVASSVPLLL FPQGFLKSIT YAI IASVML AAILSITVLA AALAILGPRV DALGVTTLLK IPFLANWQFS RRIIDWFAEK TQKTKTREEV ERGFWGRLVN VVMK RPIAF AAPILVVMVL LIIPLGQLSL GGISEKYLPP DNAVRQSQEQ FDKLFPGFRT EPLTLVMKRE DGEPITDAQI ADMRA KALT VSGFTDPDND PEKMWKERPA NDSGSKDPSV RVIQNGLENR NDAAKKIDEL RALQPPHGIE VFVGGTPALE QDSIHS LFD KLPLMALILI VTTTVLMFLA FGSVVLPIKA ALMSALTLGS TMGILTWMFV DGHGSGLMNY TPQPLMAPMI GLIIAVI WG LSTDYEVFLV SRMVEARERG MSTAEAIRIG TATTGRLITG AALILAVVAG AFVFSDLVMM KYLAFGLLIA LLLDATII R MFLVPAVMKL LGDDCWWAPR WMKRVQEKLG LGETELPDER KRPTVRESET DQRENLYFQ

UniProtKB: Trehalose monomycolate exporter MmpL3

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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

Concentration3 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris pH7.5. 150 mM NaCl
Sugar embeddingMaterial: peptidiscs
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 10004 / Average exposure time: 3.5 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFalcon 4i
Particle selectionNumber selected: 1909486
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7k7m

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 348157
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7k7m


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8qkk:
Cryo-EM structure of MmpL3 from Mycobacterium smegmatis reconstituted into peptidiscs

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