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- PDB-8fee: Structure of Mce1 transporter from Mycobacterium smegmatis in the... -

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Basic information

Entry
Database: PDB / ID: 8fee
TitleStructure of Mce1 transporter from Mycobacterium smegmatis in the absence of LucB (Map2)
Components
  • (Virulence factor Mce family ...) x 4
  • ABC transporter, ATP-binding protein,Green fluorescent protein chimeraATP-binding cassette transporter
  • ABC-transporter integral membrane protein
  • Conserved hypothetical integral membrane protein Yrbe1a
  • MCE-family protein MCE1c
  • Mce-family protein mce1f
KeywordsMEMBRANE PROTEIN / Membrane protein complex / ABC transporter / Virulence factor / Lipid transport
Function / homology
Function and homology information


ATP-binding cassette (ABC) transporter complex / bioluminescence / generation of precursor metabolites and energy / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Virulence factor Mce protein / Mammalian cell entry, C-terminal / Cholesterol uptake porter CUP1 of Mce4, putative / ABC transporter permease MalE / Permease MlaE / Mce/MlaD / MlaD protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein ...Virulence factor Mce protein / Mammalian cell entry, C-terminal / Cholesterol uptake porter CUP1 of Mce4, putative / ABC transporter permease MalE / Permease MlaE / Mce/MlaD / MlaD protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Prokaryotic membrane lipoprotein lipid attachment site profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Unknown ligand / ABC-transporter integral membrane protein / Virulence factor Mce family protein / Virulence factor Mce family protein / Virulence factor mce family protein / Virulence factor Mce family protein / Mce-family protein mce1f / ABC transporter, ATP-binding protein / Conserved hypothetical integral membrane protein Yrbe1a / MCE-family protein MCE1c / Green fluorescent protein
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsChen, J. / Bhabha, G. / Ekiert, D.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)PEW-00033055 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nature / Year: 2023
Title: Structure of an endogenous mycobacterial MCE lipid transporter.
Authors: James Chen / Alice Fruhauf / Catherine Fan / Jackeline Ponce / Beatrix Ueberheide / Gira Bhabha / Damian C Ekiert /
Abstract: To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE) proteins are important virulence ...To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids and cholesterol across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
History
DepositionDec 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed
Revision 1.2Aug 9, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Aug 23, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Virulence factor Mce family protein
B: Virulence factor Mce family protein
C: MCE-family protein MCE1c
D: Virulence factor mce family protein
E: Virulence factor Mce family protein
F: Mce-family protein mce1f
G: ABC transporter, ATP-binding protein,Green fluorescent protein chimera
H: ABC transporter, ATP-binding protein,Green fluorescent protein chimera
I: Conserved hypothetical integral membrane protein Yrbe1a
J: ABC-transporter integral membrane protein


Theoretical massNumber of molelcules
Total (without water)492,84741
Polymers492,84710
Non-polymers031
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Virulence factor Mce family ... , 4 types, 4 molecules ABDE

#1: Protein Virulence factor Mce family protein


Mass: 43944.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR2
#2: Protein Virulence factor Mce family protein


Mass: 37467.738 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR3
#4: Protein Virulence factor mce family protein


Mass: 58054.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR5
#5: Protein Virulence factor Mce family protein


Mass: 42566.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR6

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Protein , 5 types, 6 molecules CFGHIJ

#3: Protein MCE-family protein MCE1c


Mass: 54737.805 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: I7G2J2
#6: Protein Mce-family protein mce1f


Mass: 54342.898 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR7
#7: Protein ABC transporter, ATP-binding protein,Green fluorescent protein chimera / ATP-binding cassette transporter


Mass: 71624.820 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: C-terminus of MceG is tagged (3C-eGFP-4xGly-Tev-Flag-His6)
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QS64, UniProt: P42212
#8: Protein Conserved hypothetical integral membrane protein Yrbe1a


Mass: 27674.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: I7F4Q4
#9: Protein ABC-transporter integral membrane protein


Mass: 30809.025 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QNR1

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Non-polymers , 1 types, 31 molecules

#10: Chemical...
ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 31 / Source method: obtained synthetically / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
Type: COMPLEX
Details: Complex was isolated directly from Mycobacterium smegmatis by pulling down MceG-GFP using GFP-affinity purification and size exclusion chromatography
Entity ID: #1-#9 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 7.5
Details: 50 mM Tris-HCl pH 7.5, 5 mM MgSO4, 150 mM NaCl, 1 mM DDM, 1 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClTrisC4H13Cl2NO31
25 mMMagnesium sulfateMgSO41
3150 mMSodium chlorideNaClSodium chloride1
41 mMn-dodecyl-beta-D-maltosideC24H46O111
51 mMDithiothreitolC4H10O2S21
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample contains a mixture of MCE proteins endogenously purified from Mycobacterium smegmatis.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 43925 / Details: Images were collected in super resolution mode.
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1Topaz0.2.3particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.1CTF correction
7UCSF Chimerav1.16model fitting
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC3.3.1classification
12cryoSPARC3.3.13D reconstruction
13PHENIXv1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2869223
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160443 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Model was initial fitted into the map using Chimera follow-by rigid body refinement in PHENIX. Models were further refined using PHENIX real-space refinement and then manually inspected in Coot.

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