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

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Basic information

Entry
Database: PDB / ID: 7t3n
TitleR184Q/E191Q mutant of rat vesicular glutamate transporter 2 (VGLUT2)
ComponentsVesicular glutamate transporter 2
KeywordsTRANSPORT PROTEIN / synaptic vesicle / neurotransmitter / glutamate transport
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 / potassium:proton antiporter activity / L-glutamate transmembrane transport / L-glutamate transmembrane transporter 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
(1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID / Chem-I7Y / Vesicular glutamate transporter 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsLi, F. / Stroud, R.M. / Edwards, R.H.
Funding support United States, 2items
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, R.M.
History
DepositionDec 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Vesicular glutamate transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,8743
Polymers64,8461
Non-polymers1,0282
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Vesicular glutamate transporter 2 / VGluT2 / Differentiation-associated BNPI / Differentiation-associated Na(+)-dependent inorganic ...VGluT2 / Differentiation-associated BNPI / Differentiation-associated Na(+)-dependent inorganic phosphate cotransporter / Solute carrier family 17 member 6


Mass: 64845.637 Da / Num. of mol.: 1 / Mutation: R184Q, E191Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Slc17a6, Dnpi, Vglut2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9JI12
#2: Chemical ChemComp-C5B / (1S,3R)-1-AMINOCYCLOPENTANE-1,3-DICARBOXYLIC ACID / 1S,3R-ACPD


Mass: 173.167 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C7H11NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-I7Y / (2R)-2-(methoxymethyl)-4-{[(25R)-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 855.060 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C45H74O15
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: complex of rat vesicular glutamate transporter 2 with a Fab
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal defocus max: 20000 nm / Nominal defocus min: 8000 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
7Cootmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126964 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6V4D

6v4d
PDB Unreleased entry

RefinementHighest resolution: 3 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0053617
ELECTRON MICROSCOPYf_angle_d0.6394936
ELECTRON MICROSCOPYf_dihedral_angle_d8.4512732
ELECTRON MICROSCOPYf_chiral_restr0.046564
ELECTRON MICROSCOPYf_plane_restr0.005598

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