EMDB-3356: Activation of NMDA receptors and the mechanism of inhibition by i...

Basic information

• Entry: Database: EMDB / ID: 3356
• Title: Activation of NMDA receptors and the mechanism of inhibition by ifenprodil - Class Y
• Map data: Class Y, unsharpened, unmasked map

Sample

NMDA Receptor

• (N-methyl-D-aspartate receptor ...NMDA receptor) x 2

• Keywords: NMDA receptor / glutamate receptor / GluN1 / GluN2B / ion channel
• Function / homology: Periplasmic binding protein-like I / Ionotropic glutamate receptor, metazoa / N-methyl D-aspartate receptor 2B3 C-terminus / Ligand-gated ion channel / Calmodulin-binding domain C0 of NMDA receptor NR1 subunit / Receptor family ligand binding region / EPHB-mediated forward signaling / Unblocking of NMDA receptor, glutamate binding and activation / Ionotropic glutamate receptor / Ligated ion channel L-glutamate- and glycine-binding site / CREB phosphorylation through the activation of CaMKII / Receptor, ligand binding region / Synaptic adhesion-like molecules / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / RAF/MAP kinase cascade / Glutamate [NMDA] receptor, epsilon subunit, C-terminal / Calmodulin-binding domain C0, NMDA receptor, NR1 subunit / Ras activation upon Ca2+ influx through NMDA receptor / sensitization / regulation of postsynaptic cytosolic calcium ion concentration / cellular response to curcumin / cellular response to magnesium starvation / cellular response to corticosterone stimulus / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / NMDA selective glutamate receptor signaling pathway / dendritic branch / response to other organism / calcium ion transmembrane import into cytosol / cellular response to lipid / glutamate-gated calcium ion channel activity / neurotransmitter binding / action potential / cellular response to manganese ion / cellular response to dsRNA / apical dendrite / glycine binding / voltage-gated cation channel activity / dendrite membrane / positive regulation of glutamate secretion / multicellular organismal response to stress / NMDA glutamate receptor activity / response to carbohydrate / NMDA selective glutamate receptor complex / glutamate binding / positive regulation of reactive oxygen species biosynthetic process / synaptic membrane / positive regulation of cysteine-type endopeptidase activity / response to methylmercury / response to amine / response to manganese ion / response to magnesium ion / cellular response to forskolin / positive regulation of calcium ion transport into cytosol / synaptic cleft / response to growth hormone / receptor clustering / excitatory synapse / associative learning / regulation of MAPK cascade / behavioral fear response / long-term memory / response to amphetamine / response to cocaine / positive regulation of dendritic spine maintenance / positive regulation of excitatory postsynaptic potential / phosphatase binding / positive regulation of synaptic transmission / cerebral cortex development / response to fungicide / response to organonitrogen compound / ionotropic glutamate receptor activity / extracellularly glutamate-gated ion channel activity / cellular response to growth factor stimulus / hippocampus development / cellular response to organic cyclic compound / ionotropic glutamate receptor signaling pathway / response to electrical stimulus / positive regulation of cell death / cellular response to amino acid stimulus / memory / regulation of long-term neuronal synaptic plasticity / presynaptic membrane / response to cytokine / response to toxic substance / response to mechanical stimulus / terminal bouton / response to calcium ion / Z disc / rhythmic process / response to organic cyclic compound / protein heterotetramerization / learning or memory / protein tetramerization / postsynaptic membrane / protein-containing complex binding / drug binding / postsynaptic density / neuron projection / dendritic spine / response to ethanol
• Source: Rattus norvegicus (Norway rat)
• Method: single particle reconstruction / cryo EM / 6.25 Å resolution
• Authors: Tajima N / Karakas E / Grant T / Simorowski N / Diaz-Avalos R / Grigorieff N / Furukawa H
• Citation: Journal: 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
• Validation Report: PDB-ID: 5fxk; ; SummaryFull reportAbout validation report
• Date: Deposition: Mar 2, 2016 / Header (metadata) release: Apr 6, 2016 / Map release: May 11, 2016 / Last update: Jun 15, 2016

Map

• File: emd_3356.map.gz (map file in CCP4 format, 65537 KB)
• Voxel size: X=Y=Z: 1.31 Å

Density

Contour Level:	0.038 (by author), 0.038 (movie #1):
Minimum - Maximum	-0.0173353 - 0.08615234
Average (Standard dev.)	-0.00064702 (0.0065396)

Details

EMDB XML:

Space Group Number	1
Map Geometry	Axis order X Y Z Dimensions 256 256 256 Origin 0 0 0 Limit 255 255 255 Spacing 256 256 256
Cell	A=B=C: 335.36 Å α=β=γ: 90 deg.

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.31	1.31	1.31
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	335.360	335.360	335.360
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	256	256	256
D min/max/mean	-0.017	0.086	-0.001

Supplemental data

Sample components

Entire NMDA Receptor

• Entire: Name: 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
• Mass: Theoretical: 370 kDa

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

• Protein: Name: N-methyl-D-aspartate receptor GluN1 / a.k.a: GluN1, NR1 / Oligomeric Details: dimer / Number of Copies: 2 / Recombinant expression: Yes
• Mass: Theoretical: 93 kDa
• Source: Species: 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 references: UniProt: Glutamate receptor ionotropic, NMDA 1

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

• Protein: Name: N-methyl-D-aspartate receptor GluN2B / a.k.a: GluN2B, NR2B / Oligomeric Details: Dimer / Recombinant expression: Yes / Number of Copies: 2
• Mass: Theoretical: 92 kDa
• Source: Species: 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 references: UniProt: Glutamate receptor ionotropic, NMDA 2B

Experimental details

Sample preparation

• Specimen: Specimen state: particle / Method: cryo EM
• Sample solution: Specimen 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 film: C-flat 1.2/1.3 Cu 400
• Vitrification: Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 90 % / Method: 3s Blot time

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Imaging: Microscope: 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 gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 100 e/Ų / Electron beam tilt params: 0 / Illumination mode: FLOOD BEAM
• Lens: Magnification: 22500 X (nominal), 38168 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2500 nm
• Specimen Holder: Model: FEI TITAN KRIOS AUTOGRID HOLDER
• Camera: Detector: GATAN K2 SUMMIT (4k x 4k)

Image acquisition

• Image acquisition: Number of digital images: 1200

Image processing

• Processing: Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 15000
• 3D reconstruction: Algorithm: FREALIGN / Software: Unblur, CTFFIND4, FREALIGN / CTF correction: Each Particle; Details: The highest resolution included in the refinement was 8A.; Resolution: 6.25 Å / Resolution method: FSC 0.143, semi-independent

Atomic model buiding

Modeling #1

Software: 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

4pe5

Chain ID: A, B, C, D

Output model

5fxk