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- PDB-8x0c: Human FL Metabotropic glutamate receptor 5, mGlu5-5M with quisqua... -

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Basic information

Entry
Database: PDB / ID: 8x0c
TitleHuman FL Metabotropic glutamate receptor 5, mGlu5-5M with quisqualate and VU0424465, conformer 1
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 / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / 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) ...A2A adenosine receptor binding / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / 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 / postsynaptic density membrane / G protein-coupled receptor activity / Schaffer collateral - CA1 synapse / cognition / cellular response to amyloid-beta / chemical synaptic transmission / G alpha (q) signalling events / dendritic spine / learning or memory / positive regulation of MAPK cascade / 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 / G-protein coupled receptors family 3 signature 3. / GPCR, family 3, conserved site / 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: 3.2 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)188,8568
Polymers187,3832
Non-polymers1,4736
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: 93691.492 Da / Num. of mol.: 2 / Mutation: T742A,S753A,T777A,I799A,A813L,N455A,H350L
Source method: isolated from a genetically manipulated source
Details: The construct has tags and protease cleavage site at its N-term (DYKDDDDKHHHHHHHHHHLEVLFQGP) and when compared to uniprot it starts at 21 and ends at 856. For CryoEM, the tag is cleaved and ...Details: The construct has tags and protease cleavage site at its N-term (DYKDDDDKHHHHHHHHHHLEVLFQGP) and when compared to uniprot it starts at 21 and ends at 856. For CryoEM, the tag is cleaved and the protein used for experiment starts at QSSE but residues starting RR have been modeled. There are 5 thermostabilising mutations (T742A, S753A, T777A, I799A, A813L). Residue N445 is mutated to remove glycosylation. An additional mutation H350L is engineered for a nanobody to bind. Each monomer has one sugar modelled and 10 disulfide bonds.
Source: (gene. exp.) Homo sapiens (human) / Gene: GRM5 / Plasmid: BacMam / Cell line (production host): HEK293 / 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
#4: Chemical ChemComp-XQT / VU0424465 / 5-[2-(3-fluorophenyl)ethynyl]-~{N}-[(2~{R})-3-methyl-3-oxidanyl-butan-2-yl]pyridine-2-carboxamide


Mass: 326.365 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H19FN2O2 / Feature type: SUBJECT OF INVESTIGATION
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: Human FL Metabotropic glutamate receptor 5, mGlu5-5M with Quiaqualate and VU0424465
Type: COMPLEX
Details: Receptor is purified in detergent micelles and monodisperse. The receptor is purified with 10 uM Quisqualate and 10 uM 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) / Cell: HEK293S GnTI- / Plasmid: BacMam
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMsodium chloride1
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 R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Blotting time was 5 seconds, blot force of 10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Data was collected with fringe-free imaging and aberration-free image shift. Selectris detector was used in EFTEM mode.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 192572 X / Nominal defocus max: 1200 nm / Nominal defocus min: 500 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: 4.9 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 19115 / Details: Electron flux was 5.4 /e/A2/s. No. of frames 1566
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1.14CTF correction
5RELION4CTF correction
6cryoSPARC4.3.0CTF correction
9UCSF Chimera1.15model fitting
10Coot0.9.8.7model fitting
13cryoSPARCfinal 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: 4481289
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92933 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 38.9 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingAccession code: D_1300042377 / Details: This model is derived from the deposition ID / Source name: Other / Type: other
RefinementResolution: 3.2→184.07 Å / Cor.coef. Fo:Fc: 0.865 / SU B: 28.485 / SU ML: 0.487 / ESU R: 0.597
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.40401 --
obs0.40401 105823 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 38.444 Å2
Refinement stepCycle: 1 / Total: 12180
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01212480
ELECTRON MICROSCOPYr_bond_other_d00.01611838
ELECTRON MICROSCOPYr_angle_refined_deg0.9341.6416930
ELECTRON MICROSCOPYr_angle_other_deg0.3371.56427260
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.80151530
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2.5915.28670
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.368102130
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0480.21894
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0214306
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022866
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_it0.0973.8946138
ELECTRON MICROSCOPYr_mcbond_other0.0973.8946138
ELECTRON MICROSCOPYr_mcangle_it0.1826.9987662
ELECTRON MICROSCOPYr_mcangle_other0.1826.9987663
ELECTRON MICROSCOPYr_scbond_it0.0613.8976342
ELECTRON MICROSCOPYr_scbond_other0.0613.8976343
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other0.1247.2119269
ELECTRON MICROSCOPYr_long_range_B_refined0.58946.251295
ELECTRON MICROSCOPYr_long_range_B_other0.58946.251296
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.2→3.283 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.757 7916 -
obs--100 %

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