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- EMDB-25665: R184Q/E191Q mutant of rat vesicular glutamate transporter 2 (VGLUT2) -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-25665
TitleR184Q/E191Q mutant of rat vesicular glutamate transporter 2 (VGLUT2)
Map dataSharpened map for model building
Sample
  • Complex: complex of rat vesicular glutamate transporter 2 with a Fab
    • Protein or peptide: Vesicular glutamate transporter 2
  • Ligand: (1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID
  • Ligand: (2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside
Function / homology
Function and homology information


sodium:phosphate symporter activity / sodium-dependent phosphate transport / phosphate ion uniporter activity / L-glutamate uniporter activity / pericellular basket / Organic anion transporters / neurotransmitter uptake / neurotransmitter loading into synaptic vesicle / hyaloid vascular plexus regression / L-glutamate import ...sodium:phosphate symporter activity / sodium-dependent phosphate transport / phosphate ion uniporter activity / L-glutamate uniporter activity / pericellular basket / Organic anion transporters / neurotransmitter uptake / neurotransmitter loading into synaptic vesicle / hyaloid vascular plexus regression / L-glutamate import / phosphate ion transport / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / potassium:proton antiporter activity / neurotransmitter transmembrane transporter activity / phosphate ion homeostasis / neural retina development / regulation of synapse structure or activity / monoatomic anion transport / chloride channel activity / chloride channel complex / excitatory synapse / synaptic transmission, glutamatergic / hippocampus development / synaptic vesicle membrane / synaptic vesicle / presynapse / early endosome / neuron projection / plasma 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
Vesicular glutamate transporter 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLi F / Stroud RM / Edwards RH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS089713 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)K99MH119591 United States
CitationJournal: To Be Published
Title: Substrate Recognition, Directional Flux, and Allostery in a Synaptic Vesicle Glutamate Transporter
Authors: Li F / Stroud RM
History
DepositionDec 8, 2021-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateDec 14, 2022-
Current statusDec 14, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25665.png

Downloads & links

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Map

FileDownload / File: emd_25665.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map for model building
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 6.1
Minimum - Maximum-35.415752 - 59.031727
Average (Standard dev.)-1.912284e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 334.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : complex of rat vesicular glutamate transporter 2 with a Fab

EntireName: complex of rat vesicular glutamate transporter 2 with a Fab
Components
  • Complex: complex of rat vesicular glutamate transporter 2 with a Fab
    • Protein or peptide: Vesicular glutamate transporter 2
  • Ligand: (1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID
  • Ligand: (2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside

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Supramolecule #1: complex of rat vesicular glutamate transporter 2 with a Fab

SupramoleculeName: complex of rat vesicular glutamate transporter 2 with a Fab
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Vesicular glutamate transporter 2

MacromoleculeName: Vesicular glutamate transporter 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 64.845637 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MESVKQRILA PGKEGIKNFA GKSLGQIYRV LEKKQDNRET IELTEDGKPL EVPEKKAPLC D(P1L)TCFGLPRR YIIAIM SGL GFCISFGIRC NLGVAIVDMV NNSTIHRGGK VIKEKAKFNW DPETVGMIHG SFFWGYIITQ IPGGYIASRL AANRVFG AA ILLTSTLNML ...String:
MESVKQRILA PGKEGIKNFA GKSLGQIYRV LEKKQDNRET IELTEDGKPL EVPEKKAPLC D(P1L)TCFGLPRR YIIAIM SGL GFCISFGIRC NLGVAIVDMV NNSTIHRGGK VIKEKAKFNW DPETVGMIHG SFFWGYIITQ IPGGYIASRL AANRVFG AA ILLTSTLNML IPSAARVHYG CVIFVQILQG LVQGVTYPAC HGIWSKWAPP LERSRLATTS FCGSYAGAVI AMPLAGIL V QYTGWSSVFY VYGSFGMVWY MFWLLVSYES PAKHPTITDE ERRYIEESIG ESANLLGAME KFKTPWRKFF TSMPVYAII VANFCRSWTF YLLLISQPAY FEEVFGFEIS KVGMLSAVPH LVMTIIVPIG GQIADFLRSK QILSTTTVRK IMNCGGFGME ATLLLVVGY SHTRGVAISF LVLAVGFSGF AISGFNVNHL DIAPRYASIL MGISNGVGTL SGMVCPIIVG AMTKNKSREE W QYVFLIAA LVHYGGVIFY ALFASGEKQP WADPEETSEE KCGFIHEDEL DEETGDITQN YINYGTTKSY GATSQENGGW PN GWEKKEE FVQESAQDAY SYKDRDDYS

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Macromolecule #2: (1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID

MacromoleculeName: (1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID / type: ligand / ID: 2 / Number of copies: 1 / Formula: C5B
Molecular weightTheoretical: 173.167 Da
Chemical component information

ChemComp-C5B:
(1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID

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Macromolecule #3: (2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl ...

MacromoleculeName: (2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside
type: ligand / ID: 3 / Number of copies: 1 / Formula: I7Y
Molecular weightTheoretical: 855.06 Da
Chemical component information

ChemComp-I7Y:
(2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside

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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

Concentration0.5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 20.0 µm / Nominal defocus min: 8.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6v4d
PDB Unreleased entry

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 126964
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6v4d
PDB Unreleased entry

Output model

PDB-7t3n:
R184Q/E191Q mutant of rat vesicular glutamate transporter 2 (VGLUT2)

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