Raw, consensus refinement of Msmeg Mce1 transporter (Map0e)
Map data
Raw map used as template to generate composite Map0. Map was generated by aligning consensus set of particles that were retracted with a larger boxsize (640 px). Mask was used to exclude LucB.
Sample
Complex: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
Protein or peptide: Virulence factor Mce family protein Mce1A
Protein or peptide: Virulence factor Mce family protein Mce1B
Protein or peptide: Virulence factor Mce family protein Mce1C
Protein or peptide: Virulence factor Mce family protein Mce1D
Protein or peptide: Virulence factor Mce family protein Mce1E
Protein or peptide: Virulence factor Mce family protein Mce1F
Protein or peptide: ATP-binding protein MceG
Protein or peptide: ATP-binding protein MceG
Protein or peptide: ABC transporter permease protein YrbE1A
Protein or peptide: ABC transporter permease protein YrbE1B
Keywords
Membrane protein complex / ABC transporter / Virulence factor / Lipid transport / MEMBRANE PROTEIN
Biological species
Mycolicibacterium smegmatis MC2 155 (bacteria)
Method
single particle reconstruction / cryo EM / Resolution: 2.92 Å
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
Citation
Journal: 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.
Download / File: emd_29232.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Raw map used as template to generate composite Map0. Map was generated by aligning consensus set of particles that were retracted with a larger boxsize (640 px). Mask was used to exclude LucB.
Voxel size
X=Y=Z: 0.8255 Å
Density
Contour Level
By AUTHOR: 0.15
Minimum - Maximum
-0.7296897 - 1.2698982
Average (Standard dev.)
0.000005381307 (±0.018287499)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
640
640
640
Spacing
640
640
640
Cell
A=B=C: 528.32 Å α=β=γ: 90.0 °
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Supplemental data
-
Half map: Half map A for raw map used as template to generate composite map.
Name: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG) type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Complex was isolated directly from Mycobacterium smegmatis by pulling down MceG-GFP using GFP-affinity purification and size exclusion chromatography.
Details: 50 mM Tris-HCl pH 7.5, 5 mM MgSO4, 150 mM NaCl, 1 mM DDM, 1 mM DTT
Grid
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Details
This sample contains a mixture of MCE proteins endogenously purified from Mycobacterium smegmatis.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 2 / Number real images: 43925 / Average exposure time: 2.0 sec. / Average electron dose: 60.0 e/Å2 Details: Images were collected in super resolution mode (nominal pixel size of 0.8255 A/pix after binning by 2).
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type of model: OTHER Details: Starting model was generated using cryoSPARC Ab initio Reconstruction.
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 305172
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)
-
Atomic model buiding 1
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.
Refinement
Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
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