EMDB-27149: Zebrafish MFSD2A isoform B in inward open ligand-free conformation

Basic information

Entry

Database: EMDB / ID: EMD-27149

• Title: Zebrafish MFSD2A isoform B in inward open ligand-free conformation
• Map data: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution.

Sample

• Complex: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution
  • Protein or peptide: Sodium-dependent lysophosphatidylcholine symporter 1-B
  • Protein or peptide: FAB light chain
  • Protein or peptide: FAB heavy chain
• Ligand: DODECYL-BETA-D-MALTOSIDE
• Ligand: water

• Keywords: omega-3 fatty acid / lipid transport / membrane protein / Fab

Function / homology

Function:

Synthesis of PC / fatty acid transmembrane transporter activity / lysophospholipid translocation / lysophospholipid:sodium symporter activity / lysophospholipid transport / lipid transport across blood-brain barrier / establishment of blood-brain barrier / symporter activity / transcytosis / carbohydrate transport / fatty acid transport / endoplasmic reticulum membrane / plasma membrane

Domain/homology:

Lactose permease-like / MFS/sugar transport protein / MFS transporter superfamily

Component:

Sodium-dependent lysophosphatidylcholine symporter 1-B

• Biological species: Danio rerio (zebrafish) / Mus musculus (house mouse)
• Method: single particle reconstruction / cryo EM / Resolution: 3.4 Å
• Authors: Nguyen C / Lei HT / Lai LTF / Gallentino MJ / Mu X / Matthies D / Gonen T

Funding support

United States, 3 items

Organization	Grant number	Country
Howard Hughes Medical Institute (HHMI)		United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	P41-GM136508	United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)	ZIA HD008998-01	United States

• Citation: Journal: Nat Commun / Year: 2023; Title: Lipid flipping in the omega-3 fatty-acid transporter.; Authors: Chi Nguyen / Hsiang-Ting Lei / Louis Tung Faat Lai / Marc J Gallenito / Xuelang Mu / Doreen Matthies / Tamir Gonen / ; Abstract: Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the recently determined structures of Mfsd2a, the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.

History

Deposition	May 30, 2022	-
Header (metadata) release	May 10, 2023	-
Map release	May 10, 2023	-
Update	Nov 20, 2024	-
Current status	Nov 20, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_27149.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution.
• Voxel size: X=Y=Z: 0.844 Å

Density

Contour Level	By AUTHOR: 0.6
Minimum - Maximum	-2.4154344 - 3.7719104
Average (Standard dev.)	0.0027539537 (±0.04189093)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 384 384 384 Spacing 384 384 384
Cell	A=B=C: 324.096 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Single-particle cryo-EM map of MFSD2A isoform B from...

• File: emd_27149_additional_1.map
• Annotation: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, unsharpened.

Additional map: Single-particle cryo-EM map of MFSD2A isoform B from...

• File: emd_27149_additional_2.map
• Annotation: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, sharpened B-114.

Half map: Single-particle cryo-EM half map A of MFSD2A isoform...

• File: emd_27149_half_map_1.map
• Annotation: Single-particle cryo-EM half map A of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation.

Half map: Single-particle cryo-EM half map B of MFSD2A isoform...

• File: emd_27149_half_map_2.map
• Annotation: Single-particle cryo-EM half map B of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation.

Sample components

Entire : Single-particle cryo-EM map of MFSD2A isoform B from zebrafish wi...

• Entire: Name: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution

Components

• Complex: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution
  • Protein or peptide: Sodium-dependent lysophosphatidylcholine symporter 1-B
  • Protein or peptide: FAB light chain
  • Protein or peptide: FAB heavy chain
• Ligand: DODECYL-BETA-D-MALTOSIDE
• Ligand: water

Supramolecule #1: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish wi...

• Supramolecule: Name: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand-free conformation at an average resolution of 3.4 A, filtered to local resolution; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
• Source (natural): Organism: Danio rerio (zebrafish)
• Molecular weight: Theoretical: 56 KDa

Macromolecule #1: Sodium-dependent lysophosphatidylcholine symporter 1-B

• Macromolecule: Name: Sodium-dependent lysophosphatidylcholine symporter 1-B; type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Danio rerio (zebrafish)
• Molecular weight: Theoretical: 56.683965 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

MHHHHHHHHH HENLYFQGGS DEVKLAKHET KSRLSVCSKL CYAIGGAPYQ ITGCAIGFFL QIYLLDVALL DPFYASIILF VGRAWDAVT DPTVGFLVSR TPWTRFGRMM PWIVLSTPFA VLCYFLIWYV PSVDQGKVVW YLIFYCCFQT LQTCFHVPYS A LTMFISTE QKERDSATAY RMTVEVLGTL IGTAIQGQIV GMANAPCIST EIDLQSTGLE VAPDVQITDP HVSLQDLRNA YM IASGVIC AIYVVCAVVL FLGVKEQKDT CRVRTEPMSF FQGICMVMGH GPYAKLVMGF LFTSLAFMLL EGNFALFCIY NLG FRNDFQ NVLLVIMLSA TLAIPFWQWF LTKFGKKTAV YIGTTSVVPF LISVVLVPSS LAVTYIASFA AGVSVAAAFL LPWS MLPDV VDDFKVQNPE SQGHEAIFYS FYVFFTKFAS GVSLGVSTLS LDFAGYVTRG CTQPGEVKLT LKILVSAAPI VLIII GLLI FISYPINEEK RQGNRKLLNE QREQ

UniProtKB: Sodium-dependent lysophosphatidylcholine symporter 1-B

Macromolecule #2: FAB light chain

• Macromolecule: Name: FAB light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Mus musculus (house mouse)
• Molecular weight: Theoretical: 20.674949 KDa
• Recombinant expression: Organism: Mus musculus (house mouse)

Sequence

String:

ALDINSPEAE KNAKGARARI TCNAGNQVGS AVAWFNQRPG DPASLLTYWA ATEKGVAGKQ SAQGASTKFS MSSAGPEAPS LSSYWCLLF EKGAFSFGGS KLNPREGAGP QASILPPSAD LNTSGGAAVV CFLPNWYGNI TVQWKTEAPQ SQANMSWPGQ A GANAAYAM AAVLAITKGD YGPGSFTCNA SNRGTGPFAM SLN

Macromolecule #3: FAB heavy chain

• Macromolecule: Name: FAB heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Mus musculus (house mouse)
• Molecular weight: Theoretical: 20.568018 KDa
• Recombinant expression: Organism: Mus musculus (house mouse)

Sequence

String:

ASKLELSGPA EPRGSKSAQI TCKAKGFPEA RFWVFWLFQR AAALDWPAAN FSGGPVQFES RFQGNASLKG SQAQANAELN IGALGSSTA TYRCGWKLAN GGFFPSWGGA NVNGAAGAKA PAVYPVEISG AGTGSVTLGC LVKGYNAKPN LTWPGASGAL T FPSELNGA LWNLASAVTG SGFPSATCAV GFGAATDVDK KVAAA

Macromolecule #4: DODECYL-BETA-D-MALTOSIDE

• Macromolecule: Name: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 4 / Number of copies: 10 / Formula: LMT
• Molecular weight: Theoretical: 510.615 Da

Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

Macromolecule #5: water

• Macromolecule: Name: water / type: ligand / ID: 5 / Number of copies: 4 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 3 mg/mL

Buffer

pH: 8
Component:

Concentration	Name
50.0 mM	HEPES
150.0 mM	NaCl
0.02 %	DDM

Details: 50 mM HEPES (pH 8), 150 mM NaCl and 0.02% DDM

• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 86 % / Chamber temperature: 277 K / Instrument: LEICA EM GP; Details: 400-mesh 1.2/1.3 Cu grids (Quantifoil) were made hydrophilic by glow discharging for two times 60 seconds with a current of 15 mA in a PELCO easiGlow system. The cryo grids were produced using a Leica EM GP (Leica). The chamber was kept at 4 C and 95% humidity (86-91% measured). 3 microliter sample at 3 mg/ml was applied to a glow-discharged holey grid, blotted for 6 s, and plunge frozen into liquid ethane and stored in liquid nitrogen..
• Details: Purified 15A9 FAB fragment was incubated overnight at 4 C with MFSD2A at a 5:1 molar ratio. DrMFSD2A-15A9 complex was injected into the size exclusion column Superdex200 to separate the unbound FAB. The size exclusion column step was also used to exchange the buffer of the MFSD2A-FAB complex into 50 mM HEPES, 150 mM NaCl and 0.02% DDM. SDS-PAGE was used to pool peak fractions containing both MFSD2A and FAB, indicating complex formation. The pooled fractions containing MFSD2A-FAB were concentrated to 3 mg/ml using Amicon spin concentrator with a 30 kDa cutoff.

Electron microscopy

• Microscope: TFS KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Details: Cryo-EM grids were loaded into a 300 keV FEI Titan Krios cryo electron microscope (ThermoFisher Scientific, formerly FEI) at HHMI Janelia Reasearch Campus, Janelia Krios 1, equipped with a Cs corrector, and Gatan energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px (superresolution pixel size 0.422 A/px) in CDS mode at a dose rate of 9.5 e-/px/s (~7.5 e-/px/s on the camera through the sample) and a defocus range of -0.5 to -1.8 micrometer using SerialEM. In total, 2,653 and 8,443 movies were collected in two separate imaging sessions, with the first dataset on a 400-mesh copper grid and the second on a 300-mesh UltrAuFoil gold grid.
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 11096 / Average exposure time: 3.75 sec. / Average electron dose: 50.0 e/Ų; Details: Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px (superresolution pixel size 0.422 A/px) in CDS mode at a dose rate of 9.5 e-/px/s (~7.5 e-/px/s on the camera through the sample) and a defocus range of -0.5 to -1.8 mircometer using SerialEM. In total, 2,653 and 8,443 movies were collected in two separate imaging sessions, with the first dataset on a 400-mesh copper grid and the second on a 300-mesh UltrAuFoil gold grid.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: Movies of 50 frames with 1 e-/A2 per frame (50 e-/A2 total dose) were automatically recorded at a nominal magnification of 81,000x, corresponding to a physical pixel size of 0.844 A/px (superresolution pixel size 0.422 A/px) in CDS mode at a dose rate of 9.5 e-/px/s (~7.5 e-/px/s on the camera through the sample) and a defocus range of -0.5 to -1.8 mircometer using SerialEM. In total, 2,653 and 8,443 movies were collected in two separate imaging sessions, with the first dataset on a 400-mesh copper grid and the second on a 300-mesh UltrAuFoil gold grid.
• Particle selection: Number selected: 3657332 ; Details: Good 2D classes generated from ~1,000 manually picked particles served as templates for automatic particle picking in RELION, resulting in 623,644 and 3,033,688 particles in dataset 1 and dataset 2 respectively
• Startup model: Type of model: INSILICO MODEL; In silico model: 513,971 particles from dataset 1 were subjected to 3D initial model building in RELION (K=2, mask =180 Angstrom).
• Final reconstruction: Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Number images used: 65517
• Initial angle assignment: Type: PROJECTION MATCHING; Software: (Name: RELION (ver. 3.1.2), cryoSPARC (ver. v3.3.2)); Details: SGD (stochastic gradient descent)
• Final angle assignment: Type: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. v3.3.2); Details: Individual ligand densities were segmented and extracted with UCSF Chimera. Extracted ligand densities were then subtracted from the MFSD2A-FAB map, generating references with (1)3 ligands bound, (2) no ligand bound, (3) only ligand 1 bound, (4) only ligand 2 bound, and (5) only ligand 3 bound, respectively. These maps served as references for 3D classification without alignment, with a tight mask covering ligand binding sites included. To avoid reference bias and overfitting, individual particle subsets obtained from 3D classification were subjected to ab-initio reconstruction, followed by non-uniform refinement, resulting in maps at 3.3 A, 3.4 A, 3.3 A, 3.4 A, and 3.4 A, with local resolutions to up to 2.8 A (3-ligands, lower resolution), 2.8 A (ligand-free), 2.8 A (1A), 2.9 A (2B), and 2.9 A (3C), respectively
• Final 3D classification: Number classes: 5 / Software - Name: cryoSPARC (ver. v3.3.2); Details: Individual ligand densities were segmented and extracted with UCSF Chimera. Extracted ligand densities were then subtracted from the MFSD2A-FAB map, generating references with (1)3 ligands bound, (2) no ligand bound, (3) only ligand 1 bound, (4) only ligand 2 bound, and (5) only ligand 3 bound, respectively. These maps served as references for 3D classification without alignment, with a tight mask covering ligand binding sites included. To avoid reference bias and overfitting, individual particle subsets obtained from 3D classification were subjected to ab-initio reconstruction, followed by non-uniform refinement, resulting in maps at 3.3 A, 3.4 A, 3.3 A, 3.4 A, and 3.4 A, with local resolutions to up to 2.8 A (3-ligands, lower resolution), 2.8 A (ligand-free), 2.8 A (1A), 2.9 A (2B), and 2.9 A (3C), respectively

Atomic model buiding 1

Initial model

PDB ID:

7mjs

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Refinement: Space: REAL / Protocol: RIGID BODY FIT

Output model

PDB-8d2t:
Zebrafish MFSD2A isoform B in inward open ligand-free conformation

Atomic model buiding 2

Initial model

PDB ID:

7mjs

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 114

Output model

PDB-8d2t:
Zebrafish MFSD2A isoform B in inward open ligand-free conformation