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- PDB-8d2u: Zebrafish MFSD2A isoform B in inward open ligand 1A conformation -

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Basic information

Entry
Database: PDB / ID: 8d2u
TitleZebrafish MFSD2A isoform B in inward open ligand 1A conformation
Components
  • FAB heavy chainFragment antigen-binding
  • FAB light chainFragment antigen-binding
  • Sodium-dependent lysophosphatidylcholine symporter 1-B
KeywordsLIPID TRANSPORT / omega-3 fatty acid / membrane protein / Fab
Function / homology
Function and homology information


Synthesis of PC / lysophospholipid:sodium symporter activity / lysophospholipid transport / lipid transport across blood-brain barrier / organic substance transport / establishment of blood-brain barrier / symporter activity / transcytosis / carbohydrate transport / fatty acid transport ...Synthesis of PC / lysophospholipid:sodium symporter activity / lysophospholipid transport / lipid transport across blood-brain barrier / organic substance transport / establishment of blood-brain barrier / symporter activity / transcytosis / carbohydrate transport / fatty acid transport / endoplasmic reticulum membrane / plasma membrane
Similarity search - Function
MFS/sugar transport protein / Lactose permease-like / MFS transporter superfamily
Similarity search - Domain/homology
Chem-ZGS / Sodium-dependent lysophosphatidylcholine symporter 1-B
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNguyen, C. / Lei, H.T. / Lai, L.T.F. / Gallentino, M.J. / Mu, X. / Matthies, D. / Gonen, T.
Funding support United States, 3items
OrganizationGrant numberCountry
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
CitationJournal: 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 ...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
DepositionMay 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sodium-dependent lysophosphatidylcholine symporter 1-B
B: FAB light chain
C: FAB heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,63821
Polymers97,9273
Non-polymers8,71118
Water905
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, size exclusion chromatography followed by SDS-PAGE, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 2 molecules BC

#2: Antibody FAB light chain / Fragment antigen-binding


Mass: 20674.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): hybridoma / Production host: Mus musculus (house mouse)
#3: Antibody FAB heavy chain / Fragment antigen-binding


Mass: 20568.018 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): hybridoma / Production host: Mus musculus (house mouse)

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Protein / Sugars , 2 types, 17 molecules A

#1: Protein Sodium-dependent lysophosphatidylcholine symporter 1-B / NLS1-B / Sodium-dependent LPC symporter 1-B / Major facilitator superfamily domain-containing protein 2A-B


Mass: 56683.965 Da / Num. of mol.: 1 / Mutation: N214Q,N225Q,N509Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: mfsd2ab, nls1b, si:ch211-194e15.3, si:ch211-210b19.5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6DEJ6
#5: Sugar
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 3 types, 7 molecules

#4: Chemical 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 / LysoPC(18:3(9Z,12Z,15Z))


Mass: 518.644 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H49NO7P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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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: Single-particle cryo-EM map of MFSD2A isoform B from zebrafish with a Fab in the inward open ligand 1A conformation at an average resolution of 3.3 A, filtered to local resolution
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.056 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Danio rerio (zebrafish)7955
31Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Mus musculus (house mouse)10090
Buffer solutionpH: 8 / Details: 50 mM HEPES (pH 8), 150 mM NaCl and 0.02% DDM
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2150 mMNaClSodium chloride1
30.02 %DDM1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 86 % / Chamber temperature: 277 K
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 ...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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
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 ...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.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.75 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11096
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 ...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.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryFitting-ID
2RELION3.1.2particle selection
8UCSF Chimeramodel fitting1
10SerialEMimage acquisition
11Gctf1.18CTF correction
13RELION3.1.2initial Euler assignment
14cryoSPARCv3.3.2initial Euler assignment
16cryoSPARCv3.3.2final Euler assignment
18cryoSPARCv3.3.2classification
20cryoSPARCv3.3.23D reconstruction
22Cootmodel fitting2
23Cootmodel refinement2
24PHENIXmodel refinement2
Image processingDetails: 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 ...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.
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94740 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model building
IDProtocolSpaceB value
1RIGID BODY FITREAL
2FLEXIBLE FITREAL116
Atomic model building
IDPDB-ID 3D fitting-ID
17MJS

7mjs

1
27MJS

7mjs

2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047040
ELECTRON MICROSCOPYf_angle_d0.8479571
ELECTRON MICROSCOPYf_dihedral_angle_d13.2332336
ELECTRON MICROSCOPYf_chiral_restr0.0491140
ELECTRON MICROSCOPYf_plane_restr0.0071132

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