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- PDB-3h6t: Crystal structure of the iGluR2 ligand-binding core (S1S2J-N754S)... -

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Basic information

Entry
Database: PDB / ID: 3h6t
TitleCrystal structure of the iGluR2 ligand-binding core (S1S2J-N754S) in complex with glutamate and cyclothiazide at 2.25 A resolution
ComponentsGlutamate receptor 2GRIA2
KeywordsMEMBRANE PROTEIN / AMPA receptor ligand-binding core / iGluR2 S1S2J-N754S / cyclothiazide / allosteric modulation
Function / homology
Function and homology information


spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / AMPA glutamate receptor activity / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding ...spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / AMPA glutamate receptor activity / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of synaptic transmission / glutamate-gated receptor activity / presynaptic active zone membrane / response to fungicide / cellular response to brain-derived neurotrophic factor stimulus / regulation of synaptic transmission, glutamatergic / somatodendritic compartment / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor binding / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / cytoskeletal protein binding / SNARE binding / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / dendritic shaft / synaptic membrane / synaptic transmission, glutamatergic / PDZ domain binding / postsynaptic density membrane / protein tetramerization / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / establishment of protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / cerebral cortex development / synaptic vesicle / presynapse / presynaptic membrane / signaling receptor activity / amyloid-beta binding / growth cone / perikaryon / chemical synaptic transmission / scaffold protein binding / postsynaptic membrane / dendritic spine / postsynaptic density / neuron projection / axon / neuronal cell body / dendrite / synapse / glutamatergic synapse / protein-containing complex binding / endoplasmic reticulum membrane / protein kinase binding / cell surface / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / : / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. ...Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / : / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like II / Periplasmic binding protein-like I / D-Maltodextrin-Binding Protein; domain 2 / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / CACODYLATE ION / CYCLOTHIAZIDE / GLUTAMIC ACID / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.25 Å
AuthorsHald, H. / Gajhede, M. / Kastrup, J.S.
Citation
Journal: J.Mol.Biol. / Year: 2009
Title: Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iGluR2.
Authors: Hald, H. / Ahring, P.K. / Timmermann, D.B. / Liljefors, T. / Gajhede, M. / Kastrup, J.S.
#1: Journal: Neuron / Year: 2000
Title: Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core.
Authors: Armstrong, N. / Gouaux, E.
#2: Journal: Nature / Year: 2002
Title: Mechanism of glutamate receptor desensitization.
Authors: Sun, Y. / Olson, R. / Horning, M. / Armstrong, N. / Mayer, M. / Gouaux, E.
History
DepositionApr 24, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.2Aug 2, 2017Group: Data collection / Refinement description / Source and taxonomy
Category: diffrn_source / entity_src_gen / software / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.3Oct 13, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate receptor 2
B: Glutamate receptor 2
C: Glutamate receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,01132
Polymers87,5843
Non-polymers3,42729
Water18,5731031
1
A: Glutamate receptor 2
C: Glutamate receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,53920
Polymers58,3892
Non-polymers2,15018
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Glutamate receptor 2
hetero molecules

B: Glutamate receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,94424
Polymers58,3892
Non-polymers2,55522
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,-y,z1
Unit cell
Length a, b, c (Å)114.486, 163.136, 47.354
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Glutamate receptor 2 / GRIA2 / GluR-2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / AMPA 2 / AMPA-selective glutamate receptor 2


Mass: 29194.658 Da / Num. of mol.: 3
Fragment: iGluR2-flop ligand-binding core: UNP residues 413-796
Mutation: N754S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Plasmid: pET30b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P19491

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Non-polymers , 8 types, 1060 molecules

#2: Chemical ChemComp-GLU / GLUTAMIC ACID / Glutamic acid


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C5H9NO4
#3: Chemical ChemComp-CYZ / CYCLOTHIAZIDE / 3-BICYCLO[2.2.1]HEPT-5-EN-2-YL-6-CHLORO-3,4- DIHYDRO-2H-1,2,4-BENZOTHIADIAZINE-7-SULFONAMIDE 1,1 DIOXIDE / Cyclothiazide


Mass: 389.878 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C14H16ClN3O4S2
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#8: Chemical ChemComp-CAC / CACODYLATE ION / dimethylarsinate / Cacodylic acid


Mass: 136.989 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6AsO2
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1031 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsNATIVE IGLUR2 IS A MEMBRANE PROTEIN. THE PROTEIN CRYSTALLIZED IS THE EXTRACELLULAR LIGAND-BINDING ...NATIVE IGLUR2 IS A MEMBRANE PROTEIN. THE PROTEIN CRYSTALLIZED IS THE EXTRACELLULAR LIGAND-BINDING CORE OF GLUR2. TRANSMEMBRANE REGIONS WERE GENETICALLY REMOVED AND REPLACED WITH A GLY-THR LINKER (RESIDUES 118-119). THEREFORE THE SEQUENCE MATCHES DISCONTINUOUSLY WITH THE REFERENCE DATABASE (UNP RESIDUES 413-527 AND 653-796, NUMBERING WITH SIGNAL PEPTIDE OF 21 AMINO ACIDS). THE TWO FIRST RESIDUES (GLY, ALA) ARE CLONING REMNANTS.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.52 Å3/Da / Density % sol: 51.28 %
Crystal growTemperature: 279 K / pH: 6.5
Details: 25 % PEG 4000, 0.3 M Zinc acetate, 0.1 M Cacodylate pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 279K

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Data collection

DiffractionMean temperature: 96.5 K
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I711 / Wavelength: 1.141
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Dec 14, 2005
RadiationMonochromator: SINGLE ASYMMETRICALLY CUT SI(111) CRYSTAL WITH HORIZONTAL DIFFRACTION PLANE. THE CRYSTAL IS BENDABLE FOR HORIZONTAL FOCUSING.
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.141 Å / Relative weight: 1
ReflectionResolution: 2.25→22.84 Å / Num. obs: 42613 / % possible obs: 99.3 % / Redundancy: 3.4 % / Biso Wilson estimate: 31.1 Å2 / Rmerge(I) obs: 0.084 / Rsym value: 0.084 / Net I/σ(I): 7.8
Reflection shellResolution: 2.25→2.37 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.346 / Mean I/σ(I) obs: 1.9 / Rsym value: 0.346 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å22.84 Å
Translation2.5 Å22.84 Å

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.2.25data scaling
PHASER1.3.3phasing
CNSrefinement
PDB_EXTRACT3.005data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1FTJ

1ftj


Resolution: 2.25→22.84 Å / Rfactor Rfree error: 0.005 / Occupancy max: 1 / Occupancy min: 0.5 / Data cutoff high absF: 2075455.12 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: ENGH & HUBER
RfactorNum. reflection% reflectionSelection details
Rfree0.228 2098 4.9 %RANDOM
Rwork0.182 ---
obs0.182 42576 99 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 46.16 Å2 / ksol: 0.315591 e/Å3
Displacement parametersBiso mean: 29.1 Å2
Baniso -1Baniso -2Baniso -3
1--3.964 Å20 Å20 Å2
2---0.12 Å20 Å2
3---4.084 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.29 Å0.22 Å
Luzzati d res low-5 Å
Luzzati sigma a0.28 Å0.22 Å
Refinement stepCycle: LAST / Resolution: 2.25→22.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6112 0 183 1031 7326
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.006
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.33
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.6
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d2.04
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.41.5
X-RAY DIFFRACTIONc_mcangle_it2.2562
X-RAY DIFFRACTIONc_scbond_it2.1422
X-RAY DIFFRACTIONc_scangle_it3.1232.5
LS refinement shellResolution: 2.25→2.27 Å / Rfactor Rfree error: 0.054 / Total num. of bins used: 41
RfactorNum. reflection% reflection
Rfree0.325 36 3.6 %
Rwork0.259 958 -
obs--100 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1CNS_TOPPAR:PROTEIN_REP.PARAMCNS_TOPPAR:PROTEIN.TOP
X-RAY DIFFRACTION2CNS_TOPPAR:WATER_REP.PARAMCNS_TOPPAR:WATER.TOP
X-RAY DIFFRACTION3CNS_TOPPAR:ION.PARAMCNS_TOPPAR:ION.TOP
X-RAY DIFFRACTION4CTZ_AC2_DMS2_GOL2_CAC.PARAMCTZ_AC2_DMS2_GOL2_CAC.TOP

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