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- PDB-7fd8: Thermostabilised full length human mGluR5-5M bound with L-quisqua... -

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Basic information

Entry
Database: PDB / ID: 7fd8
TitleThermostabilised full length human mGluR5-5M bound with L-quisqualic acid
ComponentsMetabotropic glutamate receptor 5
KeywordsMEMBRANE PROTEIN / G protein coupled receptors / Signal transduction / Metabotropic glutamate receptor 5 (GRM5)
Function / homology
Function and homology information


A2A adenosine receptor binding / regulation of translational elongation / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / 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 / positive regulation of long-term neuronal synaptic plasticity / : / desensitization of G protein-coupled receptor signaling pathway / G protein-coupled glutamate receptor signaling pathway ...A2A adenosine receptor binding / regulation of translational elongation / phospholipase C-activating G protein-coupled glutamate receptor signaling pathway / 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 / positive regulation of long-term neuronal synaptic plasticity / : / desensitization of G protein-coupled receptor signaling pathway / G protein-coupled glutamate receptor signaling pathway / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / astrocyte projection / Class C/3 (Metabotropic glutamate/pheromone receptors) / protein kinase C-activating G protein-coupled receptor signaling pathway / glutamate receptor activity / Neurexins and neuroligins / protein tyrosine kinase activator activity / : / positive regulation of protein tyrosine kinase activity / regulation of synaptic transmission, glutamatergic / protein tyrosine kinase binding / dendritic shaft / locomotory behavior / learning / G protein-coupled receptor activity / postsynaptic density membrane / synapse organization / regulation of protein phosphorylation / Schaffer collateral - CA1 synapse / cognition / cellular response to amyloid-beta / regulation of translation / chemical synaptic transmission / G alpha (q) signalling events / dendritic spine / positive regulation of MAPK cascade / learning or memory / glutamatergic synapse / 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 / Nine Cysteines Domain of family 3 GPCR ...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 / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR / G-protein coupled receptors family 3 profile. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
Chem-QUS / CHOLESTEROL HEMISUCCINATE / Metabotropic glutamate receptor 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsVinothkumar, K.R. / Cannone, G. / Lebon, G.
Funding support India, United Kingdom, France, 5items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Science and Engineering Research Board (SERB)RJN-094/2017 India
Medical Research Council (MRC, United Kingdom)MC-U105184322 United Kingdom
Agence Nationale de la Recherche (ANR)ANR-17-CE18-0001 France
Agence Nationale de la Recherche (ANR)ANR-18-CE11-0001 France
CitationJournal: Cell Rep / Year: 2021
Title: Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.
Authors: Chady Nasrallah / Giuseppe Cannone / Julie Briot / Karine Rottier / Alice E Berizzi / Chia-Ying Huang / Robert B Quast / Francois Hoh / Jean-Louis Banères / Fanny Malhaire / Ludovic Berto / ...Authors: Chady Nasrallah / Giuseppe Cannone / Julie Briot / Karine Rottier / Alice E Berizzi / Chia-Ying Huang / Robert B Quast / Francois Hoh / Jean-Louis Banères / Fanny Malhaire / Ludovic Berto / Anaëlle Dumazer / Joan Font-Ingles / Xavier Gómez-Santacana / Juanlo Catena / Julie Kniazeff / Cyril Goudet / Amadeu Llebaria / Jean-Philippe Pin / Kutti R Vinothkumar / Guillaume Lebon /
Abstract: Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus ...Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.
History
DepositionJul 16, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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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)195,7028
Polymers193,9082
Non-polymers1,7946
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4190 Å2
ΔGint-31 kcal/mol
Surface area70020 Å2

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Components

#1: Protein Metabotropic glutamate receptor 5 / / mGluR5


Mass: 96953.977 Da / Num. of mol.: 2 / Mutation: H350L, N445A, T742A, S753A, T777A, I799A, A813L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GRM5, GPRC1E, MGLUR5 / Cell line (production host): HEK 293 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 / N-Acetylglucosamine


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 / Quisqualic acid


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-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestY

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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 mGluR5-5M with L-quisqualic acid and PAM VU0424465, agonist bound state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.194 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.NameFormulaBuffer-ID
125 mMHepes1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 2.85 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: mGluR5 with L-quisqualic acid and ago PAM (VU0424464) purified in DDM/CHS
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: 283.15 K / Details: Blot force was 10, 3.5 seconds blotting time

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 44500 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 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: 9 sec. / Electron dose: 67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14604
Details: Dose rate was 5.894 e/p/s. Total number of frames is 48 and each frame had a dose of 1.39 e/A2
EM imaging opticsEnergyfilter name: GIF Bioquantum
Details: Imaging was done in EFTEM mode. Images were collected using beam shift in a series of 3x3 holes before stage shift. Active beam tilt compensation was turned on during the acquisition.
Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 48

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7Coot0.9.3model fitting
8UCSF Chimera1.15model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14REFMAC5.8.0267model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 655344
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118016 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 113.4 / Protocol: OTHER / Space: RECIPROCAL
Details: Refinement was performed with jelly body restraints
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
17P2L

7p2l

A1578-827
26N50

6n50

A125-510
36N51

6n51

A1510-568

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