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- EMDB-17787: 4.0 angstrom map of outward-facing MFS transporter MHAS2168, a ho... -

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Basic information

Entry
Database: EMDB / ID: EMD-17787
Title4.0 angstrom map of outward-facing MFS transporter MHAS2168, a homologue of M. tuberculosis Rv1410, in complex with a megabody
Map dataMembrane transporter MHAS2168, a homologue of M. tuberculosis Rv1410, in complex with megabody H2 consisting of nanobody H2 grafted to HopQ domain
Sample
  • Complex: MFS transporter MHAS2168 in outward-open conformation in complex with Megabody_H2
    • Protein or peptide: MFS transporter MHAS2168, a homologue of M. tuberculosis Rv1410c, in outward-open conformation
    • Protein or peptide: Megabody_H2, derived from grafting alpaca-produced Nanobody_H2 onto the HopQ adhesin domain of Helicobacter pylori
KeywordsMajor Facilitator Superfamily transporter / megabody / outward-open conformation / triacylglyceride extraction / MEMBRANE PROTEIN
Function / homology
Function and homology information


lipid transport / transmembrane transporter activity / lipid binding / plasma membrane
Similarity search - Function
Sugar transport proteins signature 1. / Sugar transporter, conserved site / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Triacylglyceride transporter MHAS_02168/C731_2106
Similarity search - Component
Biological speciesMycolicibacterium hassiacum DSM 44199 (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.99 Å
AuthorsRemm S / Gonda I / Seeger MA
Funding support Switzerland, European Union, 3 items
OrganizationGrant numberCountry
Swiss National Science FoundationPP00P3_144823 Switzerland
European Research Council (ERC)consolidator grant nr 772190European Union
University of ZurichFK-17-035 Switzerland
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410.
Authors: Sille Remm / Dario De Vecchis / Jendrik Schöppe / Cedric A J Hutter / Imre Gonda / Michael Hohl / Simon Newstead / Lars V Schäfer / Markus A Seeger /
Abstract: Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein ...Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.
History
DepositionJul 3, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateOct 25, 2023-
Current statusOct 25, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17787.png

Downloads & links

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Map

FileDownload / File: emd_17787.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMembrane transporter MHAS2168, a homologue of M. tuberculosis Rv1410, in complex with megabody H2 consisting of nanobody H2 grafted to HopQ domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 450 pix.
= 292.5 Å		0.65 Å/pix.
x 450 pix.
= 292.5 Å		0.65 Å/pix.
x 450 pix.
= 292.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.38786677 - 0.55795616
Average (Standard dev.)0.00026359543 (±0.008347081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 292.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17787_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17787_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17787_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MFS transporter MHAS2168 in outward-open conformation in complex ...

EntireName: MFS transporter MHAS2168 in outward-open conformation in complex with Megabody_H2
Components
  • Complex: MFS transporter MHAS2168 in outward-open conformation in complex with Megabody_H2
    • Protein or peptide: MFS transporter MHAS2168, a homologue of M. tuberculosis Rv1410c, in outward-open conformation
    • Protein or peptide: Megabody_H2, derived from grafting alpaca-produced Nanobody_H2 onto the HopQ adhesin domain of Helicobacter pylori

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Supramolecule #1: MFS transporter MHAS2168 in outward-open conformation in complex ...

SupramoleculeName: MFS transporter MHAS2168 in outward-open conformation in complex with Megabody_H2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium hassiacum DSM 44199 (bacteria)

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Macromolecule #1: MFS transporter MHAS2168, a homologue of M. tuberculosis Rv1410c,...

MacromoleculeName: MFS transporter MHAS2168, a homologue of M. tuberculosis Rv1410c, in outward-open conformation
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium hassiacum DSM 44199 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSAFPQTPNR LIRPRRTSRG IAISAGGLAV LLGALDTYVV VSIVTDIMRD VGIAVNQIQR VTPIITGYLL GYIAAMPLLG RASDRFGRKL LIQISLAGFA LGSVITALAT NLDVLVAGRV IQGAASGALL PVTLALAADL WATHKRAAVL GGVGAAQELG AVLGPIYGIF ...String:
MSAFPQTPNR LIRPRRTSRG IAISAGGLAV LLGALDTYVV VSIVTDIMRD VGIAVNQIQR VTPIITGYLL GYIAAMPLLG RASDRFGRKL LIQISLAGFA LGSVITALAT NLDVLVAGRV IQGAASGALL PVTLALAADL WATHKRAAVL GGVGAAQELG AVLGPIYGIF VVWLFHHWQA VFWVNVPLAL IAMVLIHISL PPRVRTEEPQ RVDVTGGLLL ALALGLATIG LYNAEPDGKQ VLPEYGPPLI IGAVIAAVAF LVWERFARTR LLDPAGVRFR PFLIALLVSL VTGGALMVTL VNVELFGQGV LGLDQDEAVF LLARFLIALP VGALLGGWIA TRVGDRAVTA VGLLIAAGGF YLIAQWPADV LESRHDLGFV SLPTLDTDLA IAGFGLGLVI APLTSAALRV VPAAQHGIAS AAVVVARMIG MLIGIAALSA WGLYRFNQYL KEQLAALPPA PADFPGGQMA GQMMRLRTAT VQAYVLQYGE IFAITAGLCV FGAVLGLFIA GRREHAEESA DAVDGVSNAR DRAPSAALEV LFQ

UniProtKB: Triacylglyceride transporter MHAS_02168/C731_2106

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Macromolecule #2: Megabody_H2, derived from grafting alpaca-produced Nanobody_H2 on...

MacromoleculeName: Megabody_H2, derived from grafting alpaca-produced Nanobody_H2 onto the HopQ adhesin domain of Helicobacter pylori
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: QVQLQESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG HLKDYIGKCD ...String:
QVQLQESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG HLKDYIGKCD ASAISSANMT MQNQKNNWGN GCAGVEETQS LLKTSAADFN NQTPQINQAQ NLANTLIQEL GNNPFRGGGK LSDTYEQLSR LLTNDNGTNS KTSAQAINQA VNNLNERAKT LAGGTTNSPA YQATLLALRS VLGLWNSMGY AVICGGYTKS PGENNQKDFH YTDENGNGTT INCGGSTNSN GTHSYNGTNT LKADKNVSLS IEQYEKIHEA YQILSKALKQ AGLAPLNSKG EKLEAHVTTS KYGSPGGSLR LSCADAGSIF NKFPMAWYRQ APGKERELVA RISSGGSTNY ADFVKGRFTI SRDNAKSTLY LQMNSLKPED TAMYYCARII NSASNIAYWG QGTRVTVSAA LEVLFQ

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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

Concentration9.4 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris base
150.0 mMNaClSodium chloride
0.03 % (w/v)C24H46O11n-dodecyl-beta-D-maltoside

Details: 20 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.03% (w/v) beta-DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: LEICA PLUNGER
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 3 / Number real images: 11713 / Average electron dose: 66.54 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4759395
Details: Particles extracted after template picking and manual inspection of particle picks
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2) / Software - details: Non-uniform refinement / Number images used: 402229
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 273034 / Software - Name: cryoSPARC (ver. v3.2) / Software - details: Heterogeneous refinement
Details: The best class had 402229 particles and worst class had 143839 particles.

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