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- PDB-8qkk: Cryo-EM structure of MmpL3 from Mycobacterium smegmatis reconstit... -

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Basic information

Entry
Database: PDB / ID: 8qkk
TitleCryo-EM structure of MmpL3 from Mycobacterium smegmatis reconstituted into peptidiscs
ComponentsTrehalose monomycolate exporter MmpL3
KeywordsMEMBRANE PROTEIN / mycobacterium / trehalose monomycolate / TMM / peptidisc / MmpL3
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsCouston, J. / Guo, Z. / Wang, K. / Gourdon, P.E. / Blaise, M.
Funding support France, Denmark, 3items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Trehalose monomycolate exporter MmpL3


Theoretical massNumber of molelcules
Total (without water)85,4651
Polymers85,4651
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Trehalose monomycolate exporter MmpL3


Mass: 85465.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: mmpL3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): C43 / Variant (production host): delta AcrB / References: UniProt: A0QP27

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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: monomer structure of MmpL3 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM Tris pH7.5. 150 mM NaCl
SpecimenConc.: 3 mg/ml / Embedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
EM embeddingMaterial: peptidiscs
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10004

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
4cryoSPARC3.3.1CTF correction
7PHENIX1.21model fitting
13PHENIX1.21model refinement
Image processingDetails: Falcon 4i
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1909486
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 348157 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 7k7m

7k7m


Pdb chain-ID: A / Accession code: 7k7m / Source name: PDB / Type: experimental model

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