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- EMDB-3352: Activation of NMDA receptors and the mechanism of inhibition by i... -

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Basic information

Entry
Database: EMDB / ID: EMD-3352
TitleActivation of NMDA receptors and the mechanism of inhibition by ifenprodil - Active confirmation
Map data
SampleNMDA Receptor
  • (N-methyl-D-aspartate receptor ...NMDA receptor) x 2
KeywordsNMDA receptor / glutamate receptor / GluN1 / GluN2B / ion channel
Function / homology
Function and homology information


EPHB-mediated forward signaling / Unblocking of NMDA receptors, glutamate binding and activation / RAF/MAP kinase cascade / Synaptic adhesion-like molecules / Assembly and cell surface presentation of NMDA receptors / cellular response to curcumin / sensitization / cellular response to corticosterone stimulus / cellular response to magnesium starvation / regulation of postsynaptic cytosolic calcium ion concentration ...EPHB-mediated forward signaling / Unblocking of NMDA receptors, glutamate binding and activation / RAF/MAP kinase cascade / Synaptic adhesion-like molecules / Assembly and cell surface presentation of NMDA receptors / cellular response to curcumin / sensitization / cellular response to corticosterone stimulus / cellular response to magnesium starvation / regulation of postsynaptic cytosolic calcium ion concentration / D2 dopamine receptor binding / NMDA selective glutamate receptor signaling pathway / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / dendritic branch / positive regulation of Schwann cell migration / response to other organism / extracellularly glutamate-gated ion channel activity / cellular response to lipid / ligand-gated ion channel activity involved in regulation of presynaptic membrane potential / glutamate-gated calcium ion channel activity / neurotransmitter binding / positive regulation of glutamate secretion / calcium ion transmembrane import into cytosol / apical dendrite / cellular response to dsRNA / action potential / glutamate receptor binding / interleukin-1 receptor binding / multicellular organismal response to stress / voltage-gated cation channel activity / parallel fiber to Purkinje cell synapse / response to carbohydrate / NMDA glutamate receptor activity / glycine binding / NMDA selective glutamate receptor complex / glutamate binding / response to manganese ion / cellular response to forskolin / response to methylmercury / dendrite membrane / positive regulation of cysteine-type endopeptidase activity / response to growth hormone / positive regulation of reactive oxygen species biosynthetic process / response to magnesium ion / synaptic membrane / response to amine / postsynaptic density membrane / positive regulation of calcium ion transport into cytosol / associative learning / behavioral fear response / receptor clustering / ionotropic glutamate receptor binding / excitatory synapse / long-term memory / excitatory postsynaptic potential / response to amphetamine / regulation of MAPK cascade / positive regulation of dendritic spine maintenance / synaptic cleft / positive regulation of excitatory postsynaptic potential / positive regulation of synaptic transmission / phosphatase binding / response to cocaine / response to fungicide / integral component of postsynaptic density membrane / long-term synaptic potentiation / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / response to organonitrogen compound / response to electrical stimulus / cellular response to organic cyclic compound / ionotropic glutamate receptor activity / positive regulation of cell death / ionotropic glutamate receptor signaling pathway / cellular response to growth factor stimulus / cellular response to amino acid stimulus / cell adhesion molecule binding / cellular response to manganese ion / hippocampal mossy fiber to CA3 synapse / response to cytokine / response to toxic substance / cerebral cortex development / memory / response to mechanical stimulus / hippocampus development / integral component of presynaptic membrane / regulation of long-term neuronal synaptic plasticity / presynaptic membrane / terminal bouton / Z disc / response to organic cyclic compound / response to calcium ion / protein heterotetramerization / rhythmic process / beta-catenin binding / protein tetramerization / scaffold protein binding / amyloid-beta binding / drug binding / response to ethanol / dendritic spine
Ionotropic glutamate receptor, metazoa / Calmodulin-binding domain C0 of NMDA receptor NR1 subunit / Calmodulin-binding domain C0, NMDA receptor, NR1 subunit / Glutamate [NMDA] receptor, epsilon subunit, C-terminal / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Periplasmic binding protein-like I / Ligand-gated ion channel / Receptor family ligand binding region / Receptor, ligand binding region / Ionotropic glutamate receptor ...Ionotropic glutamate receptor, metazoa / Calmodulin-binding domain C0 of NMDA receptor NR1 subunit / Calmodulin-binding domain C0, NMDA receptor, NR1 subunit / Glutamate [NMDA] receptor, epsilon subunit, C-terminal / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Periplasmic binding protein-like I / Ligand-gated ion channel / Receptor family ligand binding region / Receptor, ligand binding region / Ionotropic glutamate receptor / N-methyl D-aspartate receptor 2B3 C-terminus / Ligated ion channel L-glutamate- and glycine-binding site
Glutamate receptor ionotropic, NMDA 1 / Glutamate receptor ionotropic, NMDA 2B
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsTajima N / Karakas E / Grant T / Simorowski N / Diaz-Avalos R / Grigorieff N / Furukawa H
CitationJournal: Nature / Year: 2016
Title: Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.
Authors: Nami Tajima / Erkan Karakas / Timothy Grant / Noriko Simorowski / Ruben Diaz-Avalos / Nikolaus Grigorieff / Hiro Furukawa /
Abstract: The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed ...The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.
Validation ReportPDB-ID: 5fxg

SummaryFull reportAbout validation report
History
Current status-Processing site: PDBe / Status: Released
DepositionMar 1, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseMay 11, 2016-
UpdateJun 15, 2016-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5fxg
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3352.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å
1.31 Å/pix.
x 256 pix.
= 335.36 Å
1.31 Å/pix.
x 256 pix.
= 335.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.035
Minimum - Maximum-0.02085609 - 0.0761986
Average (Standard dev.)-0.00057818 (±0.00617342)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0210.076-0.001

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Supplemental data

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Sample components

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Entire NMDA Receptor

EntireName: NMDA Receptor
Details: The sample was purified in the presence of agonists Glycine and L-glutamate.
Number of components: 2
Oligomeric State: One heterotetramer of 2 GluN1 and 2 GluN2B subunits
MassTheoretical: 370 kDa

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Component #1: protein, N-methyl-D-aspartate receptor GluN1

ProteinName: N-methyl-D-aspartate receptor GluN1 / a.k.a: GluN1, NR1 / Oligomeric Details: dimer / Number of Copies: 2 / Recombinant expression: Yes
MassTheoretical: 93 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: Modified pFL and pUCDM / Cell of expression system: Sf9 / Strain: Sf9 CRL-1711
Source (natural)Location in cell: Plasma membane
External referencesUniProt: Glutamate receptor ionotropic, NMDA 1

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Component #2: protein, N-methyl-D-aspartate receptor GluN2B

ProteinName: N-methyl-D-aspartate receptor GluN2B / a.k.a: GluN2B, NR2B / Oligomeric Details: Dimer / Recombinant expression: Yes / Number of Copies: 2
MassTheoretical: 92 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: Modified pFL and pUCDM / Cell of expression system: Sf9 / Strain: Sf9 CRL-1711
Source (natural)Location in cell: Plasma membane
External referencesUniProt: Glutamate receptor ionotropic, NMDA 2B

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL
Buffer solution: 200 mM NaCl, 20 mM HEPES pH 7.0, 10 mM Glycine, 10 mM L-Glutamate, 0.002% MNG-3
pH: 7
Support filmC-flat 1.2/1.3 Cu 400
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 90 % / Method: 3s Blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Aug 10, 2015
Details: 21s exposure into 70 frames, with an exposure rate of ~8 electrons/pixel/s on the camera.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 100 e/Å2 / Electron beam tilt params: 0 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal), 38168 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1200

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 12000
3D reconstructionAlgorithm: FREALIGN / Software: Unblur, CTFFIND4, FREALIGN / CTF correction: Each Particle
Details: The highest resolution included in the refinement was 12A.
Resolution: 6.8 Å / Resolution method: FSC 0.143, semi-independent

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Atomic model buiding

Modeling #1Software: Coot / Refinement protocol: flexible / Target criteria: Real Space / Refinement space: REAL
Details: The individual domains were initially fitted using coot and real space refinement was performed using Phenix
Input PDB model: 4PE5
Chain ID: A, B, C, D
Output model

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