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- PDB-8x0e: Human FL Metabotropic glutamate receptor 5, mGlu5-5M with agonist... -

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Basic information

Entry
Database: PDB / ID: 8x0e
TitleHuman FL Metabotropic glutamate receptor 5, mGlu5-5M with agonist and PAM, W785A mutant
ComponentsMetabotropic glutamate receptor 5
KeywordsMEMBRANE PROTEIN / G-PROTEIN COUPLED RECEPTORS / SIGNAL TRANSDUCTION / METABOTROPIC GLUTAMATE RECEPTOR / Agonist / active state
Function / homology
Function and homology information


A2A adenosine receptor binding / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / positive regulation of long-term neuronal synaptic plasticity / desensitization of G protein-coupled receptor signaling pathway / G protein-coupled glutamate receptor signaling pathway / astrocyte projection ...A2A adenosine receptor binding / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / positive regulation of long-term neuronal synaptic plasticity / desensitization of G protein-coupled receptor signaling pathway / G protein-coupled glutamate receptor signaling pathway / astrocyte projection / Class C/3 (Metabotropic glutamate/pheromone receptors) / glutamate receptor activity / Neurexins and neuroligins / protein tyrosine kinase activator activity / regulation of synaptic transmission, glutamatergic / positive regulation of calcium-mediated signaling / dendritic shaft / protein tyrosine kinase binding / learning / locomotory behavior / synapse organization / G protein-coupled receptor activity / postsynaptic density membrane / Schaffer collateral - CA1 synapse / cognition / cellular response to amyloid-beta / chemical synaptic transmission / G alpha (q) signalling events / dendritic spine / positive regulation of MAPK cascade / learning or memory / neuronal cell body / dendrite / regulation of DNA-templated transcription / glutamatergic synapse / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
GPCR, family 3, metabotropic glutamate receptor 5 / Metabotropic glutamate receptor, Homer-binding domain / Homer-binding domain of metabotropic glutamate receptor / GluR_Homer-bdg / GPCR, family 3, metabotropic glutamate receptor / : / G-protein coupled receptors family 3 signature 1. / G-protein coupled receptors family 3 signature 2. / GPCR, family 3, nine cysteines domain / GPCR, family 3, nine cysteines domain superfamily ...GPCR, family 3, metabotropic glutamate receptor 5 / Metabotropic glutamate receptor, Homer-binding domain / Homer-binding domain of metabotropic glutamate receptor / GluR_Homer-bdg / GPCR, family 3, metabotropic glutamate receptor / : / G-protein coupled receptors family 3 signature 1. / G-protein coupled receptors family 3 signature 2. / GPCR, family 3, nine cysteines domain / GPCR, family 3, nine cysteines domain superfamily / Nine Cysteines Domain of family 3 GPCR / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / G-protein coupled receptors family 3 profile. / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
Chem-QUS / Metabotropic glutamate receptor 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsVinothkumar, K.R. / Lebon, G. / Cannone, G.
Funding support India, France, 3items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/204 India
Other governmentRTI4006 India
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0019 France
CitationJournal: Nat Commun / Year: 2025
Title: Conformational diversity in class C GPCR positive allosteric modulation.
Authors: Giuseppe Cannone / Ludovic Berto / Fanny Malhaire / Gavin Ferguson / Aurelien Fouillen / Stéphanie Balor / Joan Font-Ingles / Amadeu Llebaria / Cyril Goudet / Abhay Kotecha / Vinothkumar K ...Authors: Giuseppe Cannone / Ludovic Berto / Fanny Malhaire / Gavin Ferguson / Aurelien Fouillen / Stéphanie Balor / Joan Font-Ingles / Amadeu Llebaria / Cyril Goudet / Abhay Kotecha / Vinothkumar K R / Guillaume Lebon /
Abstract: The metabotropic glutamate receptors (mGlus) are class C G protein-coupled receptors (GPCR) that form obligate dimers activated by the major excitatory neurotransmitter L-glutamate. The architecture ...The metabotropic glutamate receptors (mGlus) are class C G protein-coupled receptors (GPCR) that form obligate dimers activated by the major excitatory neurotransmitter L-glutamate. The architecture of mGlu receptor comprises an extracellular Venus-Fly Trap domain (VFT) connected to the transmembrane domain (7TM) through a Cysteine-Rich Domain (CRD). The binding of L-glutamate in the VFTs and subsequent conformational change results in the signal being transmitted to the 7TM inducing G protein binding and activation. The mGlu receptors signal transduction can be allosterically potentiated by positive allosteric modulators (PAMs) binding to the 7TMs, which are of therapeutic interest in various neurological disorders. Here, we report the cryoEM structures of metabotropic glutamate receptor 5 (mGlu) purified with three chemically and pharmacologically distinct PAMs. We find that the PAMs modulate the receptor equilibrium through their different binding modes, revealing how their interactions in the 7TMs impact the mGlu receptor conformational landscape and function. In addition, we identified a PAM-free but agonist-bound intermediate state that also reveals interactions mediated by intracellular loop 2. The activation of mGlu receptor is a multi-step process in which the binding of the PAMs in the 7TM modulates the equilibrium towards the active state.
History
DepositionNov 4, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.2Jan 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Metabotropic glutamate receptor 5
B: Metabotropic glutamate receptor 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)188,0596
Polymers187,2392
Non-polymers8214
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Metabotropic glutamate receptor 5


Mass: 93619.383 Da / Num. of mol.: 2 / Mutation: T742A,S753A,T777A,I799A,A813L,W785A,H350L
Source method: isolated from a genetically manipulated source
Details: The N-terminal sequence (DYKDDDDKHHHHHHHHHHLEVLFQGP) is the tag and linker), which has been cleaved by protease before structural experiment. Here, it is included for completion. The ...Details: The N-terminal sequence (DYKDDDDKHHHHHHHHHHLEVLFQGP) is the tag and linker), which has been cleaved by protease before structural experiment. Here, it is included for completion. The sequence when compared to uniprot starts at 21 and ends at 856 (construct used for expression). The construct has the following mutations (with reference to Uniprot ID P41594) H350L - mutation for nanobody binding T742A, S753A, T777A, I799A, A813L (thermostabilising mutant) W785A - introduced mutant to test for PAM binding. There are 10 disulfide bonds per chain and 2 NAG molecules.
Source: (gene. exp.) Homo sapiens (human) / Gene: GRM5 / Cell line (production host): HEK293S GnT- / Production host: Homo sapiens (human) / References: UniProt: P41594
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-QUS / (S)-2-AMINO-3-(3,5-DIOXO-[1,2,4]OXADIAZOLIDIN-2-YL)-PROPIONIC ACID / QUISQUALATE


Mass: 189.126 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H7N3O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: agonist*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Thermostabilised full length human mGluR5, W785A mutant
Type: COMPLEX
Details: Receptor was purified with orthosteric Quisqualate and PAM VU0424465
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293S GnT-
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2150 mMSodium ChlorideNaCl1
30.03 %dodecyl maltoside1
40.006 %cholesterol hemisuccinate1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Receptor is purified in detergent micelles and monodisperse. The receptor is purified with 10 uM Quisqualate and 10 uM PAM VU0424465
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K / Details: Blot force was set to 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 28.25 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5762
Details: Images were collected in movie mode for 60 seconds and 25 frames were stored.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2EPU2.10.0.5RELimage acquisition
4CTFFIND4.1.14CTF correction
5RELION4.0.1CTF correction
6cryoSPARC4.3.0CTF correction
9UCSF Chimera1.15model fitting
10Coot0.9.8.7model fitting
12RELION4.0.1initial Euler assignment
13cryoSPARC4.3.0final Euler assignment
15cryoSPARC4.3.03D reconstruction
16PHENIX1.20.1-4487model refinement
17REFMAC5.8.0411model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 783666
Details: The particles were extract from two batches of data collection
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 188960 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 183.8 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingAccession code: D_1300042377
Details: The initial model was from this deposition ID, which has been submitted
Source name: Other / Type: experimental model
RefinementResolution: 3.4→177.62 Å / Cor.coef. Fo:Fc: 0.977 / SU B: 11.347 / SU ML: 0.18 / ESU R: 0.261
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.2775 --
obs0.2775 206241 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 182.715 Å2
Refinement stepCycle: 1 / Total: 12086
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01212380
ELECTRON MICROSCOPYr_bond_other_d00.01611760
ELECTRON MICROSCOPYr_angle_refined_deg0.8751.64116792
ELECTRON MICROSCOPYr_angle_other_deg0.3271.56427090
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.38751524
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.486566
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.5102124
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0490.21886
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0214210
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022836
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.66717.8896114
ELECTRON MICROSCOPYr_mcbond_other6.66717.8896114
ELECTRON MICROSCOPYr_mcangle_it11.43432.167632
ELECTRON MICROSCOPYr_mcangle_other11.43332.1637633
ELECTRON MICROSCOPYr_scbond_it7.18718.6946266
ELECTRON MICROSCOPYr_scbond_other7.18618.6936267
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other12.75934.3449161
ELECTRON MICROSCOPYr_long_range_B_refined23.868214.1149451
ELECTRON MICROSCOPYr_long_range_B_other23.868214.1149452
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.345 15243 -
obs--100 %

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