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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


cellular response to curcumin / sensitization / neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / cellular response to corticosterone stimulus / cellular response to magnesium starvation / fear response / regulation of postsynaptic cytosolic calcium ion concentration / NMDA selective glutamate receptor signaling pathway / D2 dopamine receptor binding / sensory organ development ...cellular response to curcumin / sensitization / neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / cellular response to corticosterone stimulus / cellular response to magnesium starvation / fear response / regulation of postsynaptic cytosolic calcium ion concentration / NMDA selective glutamate receptor signaling pathway / D2 dopamine receptor binding / sensory organ development / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / regulation of cell communication / positive regulation of Schwann cell migration / response to glycine / pons maturation / propylene metabolic process / dendritic branch / apical dendrite / protein localization to postsynaptic membrane / response to other organism / conditioned taste aversion / cellular response to lipid / regulation of respiratory gaseous exchange / olfactory learning / neurotransmitter binding / positive regulation of glutamate secretion / behavioral response to pain / glutamate-gated calcium ion channel activity / cellular response to dsRNA / action potential / calcium ion transmembrane import into cytosol / ligand-gated ion channel activity involved in regulation of presynaptic membrane potential / regulation of postsynaptic membrane potential / neuromuscular process / interleukin-1 receptor binding / voltage-gated cation channel activity / cellular response to forskolin / NMDA glutamate receptor activity / regulation of synapse assembly / NMDA selective glutamate receptor complex / glutamate binding / negative regulation of dendritic spine maintenance / startle response / parallel fiber to Purkinje cell synapse / protein heterotetramerization / response to carbohydrate / regulation of protein kinase A signaling / glycine binding / response to methylmercury / positive regulation of cysteine-type endopeptidase activity / positive regulation of reactive oxygen species biosynthetic process / response to manganese ion / regulation of neuron apoptotic process / dendrite membrane / calcium ion homeostasis / response to growth hormone / regulation of neuronal synaptic plasticity / response to amine / cation transport / response to magnesium ion / regulation of dendrite morphogenesis / regulation of MAPK cascade / positive regulation of calcium ion transport into cytosol / suckling behavior / receptor clustering / behavioral fear response / male mating behavior / regulation of axonogenesis / ionotropic glutamate receptor binding / extracellularly glutamate-gated ion channel activity / excitatory synapse / social behavior / response to morphine / associative learning / long-term memory / positive regulation of dendritic spine maintenance / glutamate receptor binding / excitatory postsynaptic potential / cation channel activity / multicellular organismal response to stress / positive regulation of excitatory postsynaptic potential / response to amphetamine / positive regulation of synaptic transmission / synaptic cleft / phosphatase binding / long-term synaptic potentiation / ionotropic glutamate receptor activity / response to cocaine / response to fungicide / integral component of postsynaptic density membrane / response to electrical stimulus / adult locomotory behavior / detection of mechanical stimulus involved in sensory perception of pain / synaptic membrane / glutamate receptor activity / prepulse inhibition / response to organonitrogen compound / calcium channel activity / cellular response to organic cyclic compound / ionotropic glutamate receptor signaling pathway
Glutamate [NMDA] receptor, epsilon subunit, C-terminal / Periplasmic binding protein-like I / Calmodulin-binding domain C0, NMDA receptor, NR1 subunit / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Receptor, ligand binding region / Ionotropic glutamate receptor, metazoa / Ionotropic glutamate receptor
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
DepositionMar 1, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseMay 11, 2016-
UpdateJun 15, 2016-
Current statusJun 15, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • 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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