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- EMDB-23883: Single-Particle Cryo-EM Structure of Major Facilitator Superfamil... -

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Basic information

Entry
Database: EMDB / ID: EMD-23883
TitleSingle-Particle Cryo-EM Structure of Major Facilitator Superfamily Domain containing 2A in complex with LPC-18:3
Map dataMFSD2A_gallus_gallus_main_map
Sample
  • Complex: Single-Particle Cryo-EM Structure of Major Facilitator Superfamily Domain containing 2A (MFSD2A) Gallus gallus in complex with LPC-18:3
    • Protein or peptide: 2AG3 Fab heavy chain
    • Protein or peptide: 2AG3 Fab light chain
    • Protein or peptide: Major Facilitator Superfamily Domain containing 2A
  • Ligand: [(2~{R})-2-oxidanyl-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (9~{Z},12~{Z},15~{Z})-octadeca-9,12,15-trienoate
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Synthesis of PC / lysophospholipid translocation / lysophosphatidylcholine flippase activity / regulation of phosphatidylethanolamine metabolic process / regulation of phosphatidylserine metabolic process / oleate transmembrane transporter activity / regulation of phosphatidylcholine metabolic process / regulation of neuron projection arborization / retina morphogenesis in camera-type eye / photoreceptor cell morphogenesis ...Synthesis of PC / lysophospholipid translocation / lysophosphatidylcholine flippase activity / regulation of phosphatidylethanolamine metabolic process / regulation of phosphatidylserine metabolic process / oleate transmembrane transporter activity / regulation of phosphatidylcholine metabolic process / regulation of neuron projection arborization / retina morphogenesis in camera-type eye / photoreceptor cell morphogenesis / lysophospholipid:sodium symporter activity / photoreceptor cell outer segment organization / lipid transport across blood-brain barrier / very-low-density lipoprotein particle assembly / organic substance transport / negative regulation of fatty acid beta-oxidation / retinal pigment epithelium development / regulation of dendrite development / positive regulation of triglyceride biosynthetic process / establishment of blood-brain barrier / transcytosis / motor behavior / maintenance of blood-brain barrier / plasma membrane => GO:0005886 / regulation of multicellular organism growth / carbohydrate transport / energy homeostasis / cellular response to starvation / hippocampus development / cognition / positive regulation of cell growth
Similarity search - Function
Lactose permease-like / MFS transporter superfamily
Similarity search - Domain/homology
Uncharacterized protein
Similarity search - Component
Biological speciesGallus gallus (chicken) / Synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsCater RJ / Chua GL / Erramilli SK / Keener JE / Choy BC / Tokarz P / Chin CF / Quek DQY / Kloss B / Pepe JG ...Cater RJ / Chua GL / Erramilli SK / Keener JE / Choy BC / Tokarz P / Chin CF / Quek DQY / Kloss B / Pepe JG / Parisi G / Wong BH / Clarke OB / Marty MT / Kossiakoff AA / Khelashvili G / Silver DL / Mancia F
Funding support United States, Singapore, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM132120 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH12564 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128624 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117372 United States
National Research Foundation (NRF, Singapore)NRF-NRFI2017-05 Singapore
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM116799 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: Nature / Year: 2021
Title: Structural basis of omega-3 fatty acid transport across the blood-brain barrier.
Authors: Rosemary J Cater / Geok Lin Chua / Satchal K Erramilli / James E Keener / Brendon C Choy / Piotr Tokarz / Cheen Fei Chin / Debra Q Y Quek / Brian Kloss / Joseph G Pepe / Giacomo Parisi / ...Authors: Rosemary J Cater / Geok Lin Chua / Satchal K Erramilli / James E Keener / Brendon C Choy / Piotr Tokarz / Cheen Fei Chin / Debra Q Y Quek / Brian Kloss / Joseph G Pepe / Giacomo Parisi / Bernice H Wong / Oliver B Clarke / Michael T Marty / Anthony A Kossiakoff / George Khelashvili / David L Silver / Filippo Mancia /
Abstract: Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources. This nutrient ...Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na-dependent manner. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.
History
DepositionApr 20, 2021-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mjs
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23883.png

Downloads & links

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Map

FileDownload / File: emd_23883.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMFSD2A_gallus_gallus_main_map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.5476536 - 1.3001281
Average (Standard dev.)-0.00036145464 (±0.022171905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z298.800298.800298.800
α/β/γ90.00090.00090.000
start NX/NY/NZ1229869
NX/NY/NZ377377377
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.5481.300-0.000

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Supplemental data

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Mask #1

Fileemd_23883_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_23883_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_23883_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Single-Particle Cryo-EM Structure of Major Facilitator Superfamil...

EntireName: Single-Particle Cryo-EM Structure of Major Facilitator Superfamily Domain containing 2A (MFSD2A) Gallus gallus in complex with LPC-18:3
Components
  • Complex: Single-Particle Cryo-EM Structure of Major Facilitator Superfamily Domain containing 2A (MFSD2A) Gallus gallus in complex with LPC-18:3
    • Protein or peptide: 2AG3 Fab heavy chain
    • Protein or peptide: 2AG3 Fab light chain
    • Protein or peptide: Major Facilitator Superfamily Domain containing 2A
  • Ligand: [(2~{R})-2-oxidanyl-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (9~{Z},12~{Z},15~{Z})-octadeca-9,12,15-trienoate
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Single-Particle Cryo-EM Structure of Major Facilitator Superfamil...

SupramoleculeName: Single-Particle Cryo-EM Structure of Major Facilitator Superfamily Domain containing 2A (MFSD2A) Gallus gallus in complex with LPC-18:3
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: 2AG3 Fab heavy chain

MacromoleculeName: 2AG3 Fab heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synthetic construct (others)
Molecular weightTheoretical: 25.646537 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NIYSSSIHWV RQAPGKGLEW VAYIYSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSLEYLYSS GYQYKWATGL DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NIYSSSIHWV RQAPGKGLEW VAYIYSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSLEYLYSS GYQYKWATGL DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HT

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Macromolecule #2: 2AG3 Fab light chain

MacromoleculeName: 2AG3 Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synthetic construct (others)
Molecular weightTheoretical: 23.655182 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSFYNQPF TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSFYNQPF TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #3: Major Facilitator Superfamily Domain containing 2A

MacromoleculeName: Major Facilitator Superfamily Domain containing 2A / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 60.789168 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAGGGGAERV RVGAAAAGLL PPSCRQPRRR ESRERLSVCS KLCYAVGGAP YQTTGCALGF FLQIYLLDVA QLDPFYASII LFVGRAWDA ITDPMVGFFI SKTPWTRFGR LMPWIIFSTP FAVISYFLIW FVPDISTGQV MWYLIFYCIF QTLVTCFHVP Y SALTMFIS ...String:
MAGGGGAERV RVGAAAAGLL PPSCRQPRRR ESRERLSVCS KLCYAVGGAP YQTTGCALGF FLQIYLLDVA QLDPFYASII LFVGRAWDA ITDPMVGFFI SKTPWTRFGR LMPWIIFSTP FAVISYFLIW FVPDISTGQV MWYLIFYCIF QTLVTCFHVP Y SALTMFIS REQSERDSAT AYRMTVEVLG TVLGTAIQGQ IVGKAVTPCI ENPPFLSETN FSVAIRNVNM THYTGSLADT RN AYMVAAG VIGGLYILCA VILSVGVREK RESSELQSDE PVSFFRGLKL VMNHGAYIKL ITGFLFTSLA FMLLEGNFAL FCT YTLGFR NEFQNILLAI MLSATLTIPF WQWFLTRFGK KTAVYVGISS AVPFLITVVV LDSNLVVTYI VAVAAGISVA AAFL LPWSM LPDVIDDFKL QHPESRGHEA IFFSFYVFFT KFTSGVSLGI STLSLDFAGY QTRGCSQPSE VNITLKLLVS AVPVG LILL GLLLFKLYPI DEEKRRENKK ALQDLREESN SSSESDSTEL ANIVENLYFQ GSHHHHHHHH HH

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Macromolecule #4: [(2~{R})-2-oxidanyl-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethox...

MacromoleculeName: [(2~{R})-2-oxidanyl-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (9~{Z},12~{Z},15~{Z})-octadeca-9,12,15-trienoate
type: ligand / ID: 4 / Number of copies: 1 / Formula: ZGS
Molecular weightTheoretical: 518.644 Da
Chemical component information

ChemComp-ZGS:
[(2~{R})-2-oxidanyl-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (9~{Z},12~{Z},15~{Z})-octadeca-9,12,15-trienoate

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration1 mg/mL
BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Average exposure time: 3.0 sec. / Average electron dose: 58.0 e/Å2

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Image processing

CTF correctionDetails: Patch CTF
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 175738
FSC plot (resolution estimation)

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