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- PDB-8x0f: Human FL Metabotropic glutamate receptor 5, mGlu5-5M with quisqua... -

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Basic information

Entry
Database: PDB / ID: 8x0f
TitleHuman FL Metabotropic glutamate receptor 5, mGlu5-5M with quisqualate and PAM VU29
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 / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / 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 / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / G protein-coupled receptor activity involved in regulation of postsynaptic membrane potential / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / 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 / protein tyrosine kinase binding / learning / dendritic shaft / locomotory behavior / G protein-coupled receptor activity / postsynaptic density membrane / synapse organization / Schaffer collateral - CA1 synapse / cognition / cellular response to amyloid-beta / chemical synaptic transmission / G alpha (q) signalling events / positive regulation of MAPK cascade / dendritic spine / learning or memory / glutamatergic synapse / dendrite / regulation of DNA-templated transcription / 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: 3.3 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: To Be Published
Title: Conformational diversity in class C GPCR positive allosteric modulation
Authors: Vinothkumar, K.R. / Lebon, G. / Cannone, G.
History
DepositionNov 4, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release

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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)188,5887
Polymers187,3832
Non-polymers1,2055
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,N445A,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): HEK293S GnTI- / 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-XRQ / VU29 / N-(1,3-Diphenyl-1H-pyrazolo-5-yl)-4-nitrobenzamide / ~{N}-(2,5-diphenylpyrazol-3-yl)-4-nitro-benzamide


Mass: 384.387 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H16N4O3 / 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 quisqualate and VU029
Type: COMPLEX / 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 %doedecyl maltoside1
40.006 %cholesterol hemisuccinate1
SpecimenConc.: 5 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 VU29
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Data was collected in EFTEM model with selectris and Falcon 4i
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
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: 4.3 sec. / Electron dose: 49.62 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9469
Details: Electron flux was 6.1. e/p/s. total number of frames per movie were 1395
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF Chimera1.15model fitting
8Coot0.9.8.7model fitting
13cryoSPARC3D reconstruction
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1418090
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113563 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 121.7 / Protocol: OTHER / Space: REAL
Atomic model buildingAccession code: D_1300042377 / Details: Model from this deposition was used as template / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612352
ELECTRON MICROSCOPYf_angle_d0.65116742
ELECTRON MICROSCOPYf_dihedral_angle_d6.1591713
ELECTRON MICROSCOPYf_chiral_restr0.0461876
ELECTRON MICROSCOPYf_plane_restr0.0042108

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