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- PDB-6n51: Metabotropic Glutamate Receptor 5 bound to L-quisqualate and Nb43 -

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Basic information

Entry
Database: PDB / ID: 6n51
TitleMetabotropic Glutamate Receptor 5 bound to L-quisqualate and Nb43
Components
  • Metabotropic glutamate receptor 5
  • Nanobody 43Single-domain antibody
KeywordsMEMBRANE PROTEIN / Cell Surface Receptor
Function / homology
Function and homology information


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 / protein tyrosine kinase activator activity / postsynaptic modulation of chemical synaptic transmission / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / G protein-coupled glutamate receptor signaling pathway / regulation of intracellular calcium activated chloride channel activity / G protein-coupled receptor activity ...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 / protein tyrosine kinase activator activity / postsynaptic modulation of chemical synaptic transmission / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / G protein-coupled glutamate receptor signaling pathway / regulation of intracellular calcium activated chloride channel activity / G protein-coupled receptor activity / calcium-mediated signaling using intracellular calcium source / regulation of synaptic transmission, glutamatergic / synapse organization / protein tyrosine kinase binding / glutamate receptor activity / cognition / locomotory behavior / regulation of protein phosphorylation / postsynaptic density membrane / positive regulation of protein tyrosine kinase activity / learning / regulation of long-term neuronal synaptic plasticity / Schaffer collateral - CA1 synapse / regulation of translation / cellular response to amyloid-beta / chemical synaptic transmission / modulation of age-related behavioral decline / learning or memory / neuron projection / G protein-coupled receptor signaling pathway / integral component of plasma membrane / identical protein binding / plasma membrane / cytoplasm
Nine Cysteines Domain of family 3 GPCR / Receptor, ligand binding region / Receptor family ligand binding region / 7 transmembrane sweet-taste receptor of 3 GCPR / GPCR, family 3, nine cysteines domain superfamily / Periplasmic binding protein-like I / Metabotropic glutamate receptor, Homer-binding domain / GPCR, family 3, conserved site / GPCR family 3, C-terminal / GPCR, family 3, nine cysteines domain ...Nine Cysteines Domain of family 3 GPCR / Receptor, ligand binding region / Receptor family ligand binding region / 7 transmembrane sweet-taste receptor of 3 GCPR / GPCR, family 3, nine cysteines domain superfamily / Periplasmic binding protein-like I / Metabotropic glutamate receptor, Homer-binding domain / GPCR, family 3, conserved site / GPCR family 3, C-terminal / GPCR, family 3, nine cysteines domain / GPCR, family 3, metabotropic glutamate receptor / GPCR, family 3, metabotropic glutamate receptor 5 / GPCR, family 3
Metabotropic glutamate receptor 5
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsKoehl, A. / Hu, H. / Feng, D. / Sun, B. / Weis, W.I. / Skiniotis, G.S. / Mathiesen, J.M. / Kobilka, B.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS092695 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS028471 United States
CitationJournal: Nature / Year: 2019
Title: Structural insights into the activation of metabotropic glutamate receptors.
Authors: Antoine Koehl / Hongli Hu / Dan Feng / Bingfa Sun / Yan Zhang / Michael J Robertson / Matthew Chu / Tong Sun Kobilka / Toon Laeremans / Jan Steyaert / Jeffrey Tarrasch / Somnath Dutta / Rasmus Fonseca / William I Weis / Jesper M Mathiesen / Georgios Skiniotis / Brian K Kobilka /
Abstract: Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich ...Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 20, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 6, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Feb 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
B: Metabotropic glutamate receptor 5
C: Nanobody 43
D: Nanobody 43
A: Metabotropic glutamate receptor 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,94210
Polymers207,6794
Non-polymers1,2636
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Proteins co-eluted as shown by SDS-PAGE
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8450 Å2
ΔGint-35 kcal/mol
Surface area87260 Å2

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Components

#1: Protein Metabotropic glutamate receptor 5 / / mGluR5


Mass: 90484.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GRM5, GPRC1E, MGLUR5 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: P41594
#2: Antibody Nanobody 43 / Single-domain antibody


Mass: 13354.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: PE_SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): DE3
#3: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
#4: 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 / Comment: agonist*YM

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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: Complex between metabotropic glutamate receptor 5 bound to L-quisqualate and Nb43
Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1100 mMSodium ChlorideNaClSodium chloride
220 mMHepesHepes
30.02 PercentGlyco-DisogeninGDN
40.002 PercentCholesteryl HemisuccinateCHS
SpecimenConc.: 12 mg/ml
Details: Sample was mono disperse as assayed by size exclusion chromatography
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: additional 0.1%OG was added to the sample right before applying sample to grid; 3.5ul sample was applied; blot for 1 second before plunging;

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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: 47169 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.12model fitting
9PHENIX1.14model refinement
10cryoSPARCinitial Euler assignment
11RELION2.1final Euler assignment
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73472 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Target criteria: Geometry

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