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- EMDB-21040: Structure of the rat vesicular glutamate transporter 2 determined... -

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Basic information

Entry
Database: EMDB / ID: EMD-21040
TitleStructure of the rat vesicular glutamate transporter 2 determined by single particle Cryo-EM
Map datasharpened map for model building not for FSC calculation. FSC curves are calculated on unsharpened maps.
Sample
  • Complex: vesicular glutamate transporter 2 in complex with a high affinity fab
    • Protein or peptide: Vesicular glutamate transporter 2
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
Vesicular glutamate transporter 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle 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, 3 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
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
DepositionNov 27, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseMay 20, 2020-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v4d
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21040.png

Downloads & links

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Map

FileDownload / File: emd_21040.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map for model building not for FSC calculation. FSC curves are calculated on unsharpened maps.
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.017
Minimum - Maximum-0.054143272 - 0.07539398
Average (Standard dev.)8.9827796e-07 (±0.0019905956)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 291.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z291.840291.840291.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0540.0750.000

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Supplemental data

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Sample components

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Entire : vesicular glutamate transporter 2 in complex with a high affinity fab

EntireName: vesicular glutamate transporter 2 in complex with a high affinity fab
Components
  • Complex: vesicular glutamate transporter 2 in complex with a high affinity fab
    • Protein or peptide: Vesicular glutamate transporter 2

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Supramolecule #1: vesicular glutamate transporter 2 in complex with a high affinity fab

SupramoleculeName: vesicular glutamate transporter 2 in complex with a high affinity fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 100 KDa

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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: 58.893008 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: METIELTEDG KPLEVPEKKA PLCDCTCFGL PRRYIIAIMS GLGFCISFGI RCNLGVAIVD MVNNSTIHRG GKVIKEKAKF NWDPETVGM IHGSFFWGYI ITQIPGGYIA SRLAANRVFG AAILLTSTLN MLIPSAARVH YGCVIFVRIL QGLVEGVTYP A CHGIWSKW ...String:
METIELTEDG KPLEVPEKKA PLCDCTCFGL PRRYIIAIMS GLGFCISFGI RCNLGVAIVD MVNNSTIHRG GKVIKEKAKF NWDPETVGM IHGSFFWGYI ITQIPGGYIA SRLAANRVFG AAILLTSTLN MLIPSAARVH YGCVIFVRIL QGLVEGVTYP A CHGIWSKW APPLERSRLA TTSFCGSYAG AVIAMPLAGI LVQYTGWSSV FYVYGSFGMV WYMFWLLVSY ESPAKHPTIT DE ERRYIEE SIGESANLLG AMEKFKTPWR KFFTSMPVYA IIVANFCRSW TFYLLLISQP AYFEEVFGFE ISKVGMLSAV PHL VMTIIV PIGGQIADFL RSKQILSTTT VRKIMNCGGF GMEATLLLVV GYSHTRGVAI SFLVLAVGFS GFAISGFNVN HLDI APRYA SILMGISNGV GTLSGMVCPI IVGAMTKNKS REEWQYVFLI AALVHYGGVI FYALFASGEK QPWADPEETS EEKCG FIHE DELDEETGDI TQNYINYGTT KSYGATGRPL EVLFQGPHHH HHHHHHH

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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 / Details: 20 mM HEPES, 150 mM NaCl, pH 7.4 and 0.01% GDN
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 4 / Number real images: 5704 / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 36000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1919729
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 243615
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8sbe:
Structure of the rat vesicular glutamate transporter 2 determined by single-particle Cryo-EM

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