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- PDB-7c7q: Cryo-EM structure of the baclofen/BHFF-bound human GABA(B) recept... -

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Basic information

Entry
Database: PDB / ID: 7c7q
TitleCryo-EM structure of the baclofen/BHFF-bound human GABA(B) receptor in active state
Components
  • Gamma-aminobutyric acid type B receptor subunit 1
  • Gamma-aminobutyric acid type B receptor subunit 2
KeywordsMEMBRANE PROTEIN / GABAB / Cryo-EM / GPCR / active / PAM
Function / homology
Function and homology information


G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / neuron-glial cell signaling / G protein-coupled receptor heterodimeric complex / G protein-coupled GABA receptor activity / GABA receptor complex / negative regulation of adenylate cyclase activity / gamma-aminobutyric acid signaling pathway / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Schaffer collateral - CA1 synapse ...G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / neuron-glial cell signaling / G protein-coupled receptor heterodimeric complex / G protein-coupled GABA receptor activity / GABA receptor complex / negative regulation of adenylate cyclase activity / gamma-aminobutyric acid signaling pathway / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Schaffer collateral - CA1 synapse / presynaptic membrane / transmembrane signaling receptor activity / postsynaptic membrane / chemical synaptic transmission / protein heterodimerization activity / neuron projection / G protein-coupled receptor signaling pathway / dendrite / integral component of plasma membrane / extracellular region / plasma membrane / cytoplasm
GPCR family 3, gamma-aminobutyric acid receptor, type B2 / GPCR family 3, GABA-B receptor / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Sushi/SCR/CCP superfamily / Periplasmic binding protein-like I / GPCR, family 3, conserved site / GPCR family 3, C-terminal / GPCR, family 3 / Sushi/SCR/CCP domain / Receptor, ligand binding region
Gamma-aminobutyric acid type B receptor subunit 2 / Gamma-aminobutyric acid type B receptor subunit 1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMao, C. / Shen, C. / Li, C. / Shen, D. / Xu, C. / Zhang, S. / Zhou, R. / Shen, Q. / Chen, L. / Jiang, Z. / Liu, J. / Zhang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81922071 China
Ministry of Science and Technology (MoST, China)2018YFA0507003 China
CitationJournal: Cell Res. / Year: 2020
Title: Cryo-EM structures of inactive and active GABA receptor.
Authors: Chunyou Mao / Cangsong Shen / Chuntao Li / Dan-Dan Shen / Chanjuan Xu / Shenglan Zhang / Rui Zhou / Qingya Shen / Li-Nan Chen / Zhinong Jiang / Jianfeng Liu / Yan Zhang /
Abstract: Metabotropic GABA G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is ...Metabotropic GABA G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABA receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of G protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of G. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABA receptor.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 26, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric acid type B receptor subunit 1
B: Gamma-aminobutyric acid type B receptor subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)188,20912
Polymers185,8952
Non-polymers2,31410
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Gamma-aminobutyric acid type B receptor subunit 1 / Gb1


Mass: 99082.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABBR1, GPRC3A / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: Q9UBS5
#2: Protein Gamma-aminobutyric acid type B receptor subunit 2 / Gb2 / G-protein coupled receptor 51 / HG20


Mass: 86812.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABBR2, GPR51, GPRC3B / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: O75899
#3: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
#4: Chemical ChemComp-FN0 / (3S)-5,7-ditert-butyl-3-oxidanyl-3-(trifluoromethyl)-1-benzofuran-2-one


Mass: 330.342 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21F3O3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-2C0 / baclofen / (3R)-4-amino-3-(4-chlorophenyl)butanoic acid


Mass: 213.661 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H12ClNO2
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: The baclofen/BHFF-bound GABAB heterodimer / Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293F
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
150 mMHEPES1
2150 mMNaClSodium chloride1
32 mMMgCl21
40.002 (w/v)%LMNG1
50.0004 (w/v)%CHS1
6100 uMbaclofen1
750 uMBHFF1
SpecimenConc.: 2 mg/ml / 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: 277 K

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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: 29000 X / Calibrated magnification: 49310 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4624
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctfv1.18CTF correction
7Coot0.89model fitting
11RELION3.0-beta2classification
12RELION3.0-beta23D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3075533
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 237606 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7C7S
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411150
ELECTRON MICROSCOPYf_angle_d0.68815133
ELECTRON MICROSCOPYf_dihedral_angle_d13.5356591
ELECTRON MICROSCOPYf_chiral_restr0.0681728
ELECTRON MICROSCOPYf_plane_restr0.0161886

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