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- EMDB-43684: Cryo-EM structure of FLVCR2 in the outward-facing state with chol... -

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Basic information

Entry
Database: EMDB / ID: EMD-43684
TitleCryo-EM structure of FLVCR2 in the outward-facing state with choline bound
Map dataFLVCR2 in the outward-facing state in complex with choline and Fab FLV23
Sample
  • Complex: FLVCR2 in the outward-facing state complexed with choline and Fab FLV23
    • Protein or peptide: Feline leukemia virus subgroup C cellular receptor 2
    • Protein or peptide: Fab FLV23 heavy chain
    • Protein or peptide: Fab FLV23 light chain
  • Ligand: CHOLINE ION
KeywordsCholine transporter / blood-brain barrier / membrane protein / MFS fold / TRANSPORT PROTEIN
Function / homology
Function and homology information


heme transmembrane transporter activity / mitochondrial membrane / heme binding / endoplasmic reticulum membrane
Similarity search - Function
: / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Feline leukemia virus subgroup C cellular receptor 2
Similarity search - Component
Biological speciesMus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsCater RJ / Mancia F
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS129105-01 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL166866-01 United States
CitationJournal: Nature / Year: 2024
Title: Structural and molecular basis of choline uptake into the brain by FLVCR2.
Authors: Rosemary J Cater / Dibyanti Mukherjee / Eva Gil-Iturbe / Satchal K Erramilli / Ting Chen / Katie Koo / Nicolás Santander / Andrew Reckers / Brian Kloss / Tomasz Gawda / Brendon C Choy / ...Authors: Rosemary J Cater / Dibyanti Mukherjee / Eva Gil-Iturbe / Satchal K Erramilli / Ting Chen / Katie Koo / Nicolás Santander / Andrew Reckers / Brian Kloss / Tomasz Gawda / Brendon C Choy / Zhening Zhang / Aditya Katewa / Amara Larpthaveesarp / Eric J Huang / Scott W J Mooney / Oliver B Clarke / Sook Wah Yee / Kathleen M Giacomini / Anthony A Kossiakoff / Matthias Quick / Thomas Arnold / Filippo Mancia /
Abstract: Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for ...Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
History
DepositionFeb 12, 2024-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43684.png

Downloads & links

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Map

FileDownload / File: emd_43684.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFLVCR2 in the outward-facing state in complex with choline and Fab FLV23
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.245
Minimum - Maximum-1.80969 - 2.1254811
Average (Standard dev.)-0.00033968608 (±0.032298487)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: FLVCR2 in the outward-facing state in complex with...

Fileemd_43684_half_map_1.map
AnnotationFLVCR2 in the outward-facing state in complex with choline and Fab FLV23 half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: FLVCR2 in the outward-facing state in complex with...

Fileemd_43684_half_map_2.map
AnnotationFLVCR2 in the outward-facing state in complex with choline and Fab FLV23 half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FLVCR2 in the outward-facing state complexed with choline and Fab...

EntireName: FLVCR2 in the outward-facing state complexed with choline and Fab FLV23
Components
  • Complex: FLVCR2 in the outward-facing state complexed with choline and Fab FLV23
    • Protein or peptide: Feline leukemia virus subgroup C cellular receptor 2
    • Protein or peptide: Fab FLV23 heavy chain
    • Protein or peptide: Fab FLV23 light chain
  • Ligand: CHOLINE ION

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Supramolecule #1: FLVCR2 in the outward-facing state complexed with choline and Fab...

SupramoleculeName: FLVCR2 in the outward-facing state complexed with choline and Fab FLV23
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Feline leukemia virus subgroup C cellular receptor 2

MacromoleculeName: Feline leukemia virus subgroup C cellular receptor 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 60.103266 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVNESLNQEE SNDRPAPESE FQMDTSYSTQ PSGSIHPSVS GHPSVSGHPS VSGHPSVSIH PSVSIDPSVS VRPSSSALPS TLAQPSGLT HHSSLVREDS VIKVSKRRWV VVLVFSCYSL CNAFQWIQYG SINNIFMNFY GVSAFAIDWL SMCYMLTYIP L LLPVAWML ...String:
MVNESLNQEE SNDRPAPESE FQMDTSYSTQ PSGSIHPSVS GHPSVSGHPS VSGHPSVSIH PSVSIDPSVS VRPSSSALPS TLAQPSGLT HHSSLVREDS VIKVSKRRWV VVLVFSCYSL CNAFQWIQYG SINNIFMNFY GVSAFAIDWL SMCYMLTYIP L LLPVAWML EKFGLRTIAI TGSALNCLGA WVKLGSLEPH LFPVTMVGQV ICSVAQVFIL GMPSRIASVW FGADEVSTAC SV AVFGNQL GIAIGFLVPP VLVPNIKDPE KLAYHISIMF YIIGGVATFL FILVIIVFKE KPKHPPSRAQ SLSYALATTD ASY LSSIVR LFKNLNFVLL VITYGLNAGA FYALSTLLNR MVILHFPGEE VNAGRIGLTI VIAGMFGAMI SGIWLDKSKT YKET TLVVY IMTLVGMVVY TFTLNLNHLW VVFITAGTLG FFMTGYLPLG FEFAVELTYP ESEGVSSGLL NVSAQVFGIV FTISQ GQII DNHGTMFGNI FLCVFLALGS ALTAFIKSDL RRQRANKDAP ETKVQEEEEE EEGSNTSKVP VVSEAHL

UniProtKB: Feline leukemia virus subgroup C cellular receptor 2

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Macromolecule #2: Fab FLV23 heavy chain

MacromoleculeName: Fab FLV23 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.772633 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NLSSYSIHWV RQAPGKGLEW VASISSYYGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSSRYEFFY SNSWWYWPAM DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NLSSYSIHWV RQAPGKGLEW VASISSYYGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSSRYEFFY SNSWWYWPAM DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HT

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Macromolecule #3: Fab FLV23 light chain

MacromoleculeName: Fab FLV23 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.357916 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYVNPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYVNPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: CHOLINE ION

MacromoleculeName: CHOLINE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CHT
Molecular weightTheoretical: 104.171 Da
Chemical component information

ChemComp-CHT:
CHOLINE ION / Choline

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 58.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 433845

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