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- PDB-7eb2: Cryo-EM structure of human GABA(B) receptor-Gi protein complex -

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Basic information

Entry
Database: PDB / ID: 7eb2
TitleCryo-EM structure of human GABA(B) receptor-Gi protein complex
Components
  • (Gamma-aminobutyric acid type B receptor subunit ...) x 2
  • (Guanine nucleotide-binding protein ...) x 3
  • ScFv16
KeywordsMEMBRANE PROTEIN / GABAB / Cryo-EM / GPCR / Gi
Function / homology
Function and homology information


G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion ...G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion / positive regulation of growth hormone secretion / extracellular matrix protein binding / GABA receptor complex / negative regulation of adenylate cyclase activity / Class C/3 (Metabotropic glutamate/pheromone receptors) / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / positive regulation of glutamate secretion / negative regulation of synaptic transmission / Adenylate cyclase inhibitory pathway / axolemma / GABA-ergic synapse / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / dendritic shaft / response to nicotine / Regulation of insulin secretion / G protein-coupled receptor binding / mitochondrial membrane / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / Schaffer collateral - CA1 synapse / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to peptide hormone / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / osteoblast differentiation / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / GDP binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / transmembrane signaling receptor activity / signaling receptor complex adaptor activity / synaptic vesicle / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / presynaptic membrane / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / cell cortex / midbody / G alpha (i) signalling events / fibroblast proliferation / chemical synaptic transmission / G alpha (s) signalling events / postsynaptic membrane / G alpha (q) signalling events / response to ethanol / Ras protein signal transduction / cell population proliferation / dendritic spine / Extra-nuclear estrogen signaling / neuron projection / cell cycle
Similarity search - Function
GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / 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 ...GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / 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. / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/SCR/CCP superfamily / Sushi/CCP/SCR domain profile. / G-protein alpha subunit, group I / Receptor, ligand binding region / Receptor family ligand binding region / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Periplasmic binding protein-like I / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
baclofen / Chem-FN0 / Gamma-aminobutyric acid type B receptor subunit 2 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Gamma-aminobutyric acid type B receptor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsShen, C. / Mao, C. / Xu, C. / Jin, N. / Zhang, H. / Shen, D. / Shen, Q. / Wang, X. / Hou, T. / Rondard, P. ...Shen, C. / Mao, C. / Xu, C. / Jin, N. / Zhang, H. / Shen, D. / Shen, Q. / Wang, X. / Hou, T. / Rondard, P. / Chen, Z. / Pin, J. / Zhang, Y. / Liu, J.
CitationJournal: Nature / Year: 2021
Title: Structural basis of GABA receptor-G protein coupling.
Authors: Cangsong Shen / Chunyou Mao / Chanjuan Xu / Nan Jin / Huibing Zhang / Dan-Dan Shen / Qingya Shen / Xiaomei Wang / Tingjun Hou / Zhong Chen / Philippe Rondard / Jean-Philippe Pin / Yan Zhang / Jianfeng Liu /
Abstract: G-protein-coupled receptors (GPCRs) have central roles in intercellular communication. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is ...G-protein-coupled receptors (GPCRs) have central roles in intercellular communication. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABA receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with G protein. We found that a single G protein interacts with the GB2 subunit of the GABA receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABA receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.
History
DepositionMar 8, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1May 12, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 7, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Y: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: ScFv16
C: Gamma-aminobutyric acid type B receptor subunit 1
D: Gamma-aminobutyric acid type B receptor subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)314,3868
Polymers313,8426
Non-polymers5442
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABY

#1: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40414.047 Da / Num. of mol.: 1 / Mutation: S47N,G203A,E245A,A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Cell (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Cell (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Cell (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Gamma-aminobutyric acid type B receptor subunit ... , 2 types, 2 molecules CD

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


Mass: 105976.297 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
#6: Protein Gamma-aminobutyric acid type B receptor subunit 2 / Gb2 / G-protein coupled receptor 51 / HG20


Mass: 91359.445 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

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Antibody , 1 types, 1 molecules N

#4: Antibody ScFv16


Mass: 28813.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Cell (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper)

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-2C0 / baclofen / (3R)-4-amino-3-(4-chlorophenyl)butanoic acid / Baclofen


Mass: 213.661 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H12ClNO2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#8: 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

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1The GABAB-Gi1 complexCOMPLEX#1-#60MULTIPLE SOURCES
2Guanine nucleotide-binding proteinCOMPLEX#1-#31RECOMBINANT
3ScFv16COMPLEX#41RECOMBINANT
4Gamma-aminobutyric acid type B receptorCOMPLEX#5-#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Trichoplusia ni (cabbage looper)7111
23Trichoplusia ni (cabbage looper)7111
34Homo sapiens (human)9606HEK 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.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 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 / Specimen holder 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: 13843
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctfv1.18CTF correction
7Coot0.89model fitting
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5889932
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 275089 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-ID
17C7Q

7c7q

1
26PT0

6pt0

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00718896
ELECTRON MICROSCOPYf_angle_d0.76325600
ELECTRON MICROSCOPYf_dihedral_angle_d14.4096867
ELECTRON MICROSCOPYf_chiral_restr0.112874
ELECTRON MICROSCOPYf_plane_restr0.0043236

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