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- PDB-8sbe: Structure of the rat vesicular glutamate transporter 2 determined... -

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Basic information

Entry
Database: PDB / ID: 8sbe
TitleStructure of the rat vesicular glutamate transporter 2 determined by single-particle Cryo-EM
ComponentsVesicular glutamate transporter 2
KeywordsMEMBRANE PROTEIN / glutamate transport / synaptic vesicle / Major facilitator Superfamily / excitatory neurotransmission
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 loading into synaptic vesicle / neurotransmitter uptake / 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 loading into synaptic vesicle / neurotransmitter uptake / hyaloid vascular plexus regression / L-glutamate import / phosphate ion transport / potassium:proton antiporter activity / L-glutamate transmembrane transport / neurotransmitter transmembrane transporter activity / L-glutamate transmembrane transporter activity / phosphate ion homeostasis / neural retina development / monoatomic anion transport / regulation of synapse structure or activity / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLi, F. / Finer-Moore, J. / Eriksen, J. / Cheng, Y. / Edwards, R. / Stroud, R.
Funding support United States, 3items
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
American Heart Association17POST33660928 United States
CitationJournal: Science / Year: 2020
Title: Ion transport and regulation in a synaptic vesicle glutamate transporter.
Authors: Fei Li / Jacob Eriksen / Janet Finer-Moore / Roger Chang / Phuong Nguyen / Alisa Bowen / Alexander Myasnikov / Zanlin Yu / David Bulkley / Yifan Cheng / Robert H Edwards / Robert M Stroud /
Abstract: Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this ...Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.
History
DepositionApr 3, 2023Deposition site: RCSB / Processing site: RCSB
SupersessionMay 3, 2023ID: 6V4D
Revision 1.0May 3, 2023Provider: 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


Theoretical massNumber of molelcules
Total (without water)58,8931
Polymers58,8931
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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: 58893.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: synonyms: VGLUT2, differentiation-associated BNPI, solute carrier family 17 member 6, Differentiation-associated NA(+)-dependent inorganic phosphate cotransporter
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Slc17a6, Dnpi, Vglut2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9JI12

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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: Vesicular glutamate transporter 2 in complex with a high-affinity Fab
Type: COMPLEX / Entity ID: all / 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5704

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1RELION2particle selection
4CTFFIND4CTF correction
10RELION2initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1919729
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 243615 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 119.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313175
ELECTRON MICROSCOPYf_angle_d0.60144341
ELECTRON MICROSCOPYf_chiral_restr0.0438502
ELECTRON MICROSCOPYf_plane_restr0.0036539
ELECTRON MICROSCOPYf_dihedral_angle_d3.9113451

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