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- PDB-6wiv: Structure of human GABA(B) receptor in an inactive state -

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Entry
Database: PDB / ID: 6wiv
TitleStructure of human GABA(B) receptor in an inactive state
Components(Gamma-aminobutyric acid type B receptor subunit ...) x 2
KeywordsMEMBRANE PROTEIN / G protein coupled receptor (GPCR) / GABA / GABA(B) receptor / Class C GPCR / Phospholipids / Inhibitory neurotransmission.
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) / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / positive regulation of glutamate secretion / negative regulation of synaptic transmission / axolemma / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / dendritic shaft / response to nicotine / mitochondrial membrane / Schaffer collateral - CA1 synapse / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / osteoblast differentiation / transmembrane signaling receptor activity / synaptic vesicle / presynaptic membrane / G alpha (i) signalling events / chemical synaptic transmission / postsynaptic membrane / response to ethanol / dendritic spine / neuron projection / G protein-coupled receptor signaling pathway / protein heterodimerization activity / negative regulation of cell population proliferation / neuronal cell body / glutamatergic synapse / endoplasmic reticulum membrane / extracellular space / plasma membrane / cytoplasm
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/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
CHOLESTEROL / Chem-U3D / Chem-U3G / Gamma-aminobutyric acid type B receptor subunit 2 / Gamma-aminobutyric acid type B receptor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsPark, J. / Fu, Z. / Frangaj, A. / Liu, J. / Mosyak, L. / Shen, T. / Slavkovich, V.N. / Ray, K.M. / Taura, J. / Cao, B. ...Park, J. / Fu, Z. / Frangaj, A. / Liu, J. / Mosyak, L. / Shen, T. / Slavkovich, V.N. / Ray, K.M. / Taura, J. / Cao, B. / Geng, Y. / Zuo, H. / Kou, Y. / Grassucci, R. / Chen, S. / Liu, Z. / Lin, X. / Williams, J.P. / Rice, W.J. / Eng, E.T. / Huang, R.K. / Soni, R.K. / Kloss, B. / Yu, Z. / Javitch, J.A. / Hendrickson, W.A. / Slesinger, P.A. / Quick, M. / Graziano, J. / Yu, H. / Fiehn, O. / Clarke, O.B. / Frank, J. / Fan, Q.R.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088454 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125801 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U2CES030158 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM116799 United States
National Institutes of Health/Office of the DirectorR01GM107462 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103310 United States
CitationJournal: Nature / Year: 2020
Title: Structure of human GABA receptor in an inactive state.
Authors: Jinseo Park / Ziao Fu / Aurel Frangaj / Jonathan Liu / Lidia Mosyak / Tong Shen / Vesna N Slavkovich / Kimberly M Ray / Jaume Taura / Baohua Cao / Yong Geng / Hao Zuo / Yongjun Kou / Robert ...Authors: Jinseo Park / Ziao Fu / Aurel Frangaj / Jonathan Liu / Lidia Mosyak / Tong Shen / Vesna N Slavkovich / Kimberly M Ray / Jaume Taura / Baohua Cao / Yong Geng / Hao Zuo / Yongjun Kou / Robert Grassucci / Shaoxia Chen / Zheng Liu / Xin Lin / Justin P Williams / William J Rice / Edward T Eng / Rick K Huang / Rajesh K Soni / Brian Kloss / Zhiheng Yu / Jonathan A Javitch / Wayne A Hendrickson / Paul A Slesinger / Matthias Quick / Joseph Graziano / Hongtao Yu / Oliver Fiehn / Oliver B Clarke / Joachim Frank / Qing R Fan /
Abstract: The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique ...The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA receptor has two subunits, GABA and GABA, that are structurally homologous but perform distinct and complementary functions. GABA recognizes orthosteric ligands, while GABA couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
History
DepositionApr 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Aug 26, 2020Group: Database references / Structure summary / Category: chem_comp / citation / citation_author
Item: _chem_comp.pdbx_synonyms / _citation.journal_volume ..._chem_comp.pdbx_synonyms / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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)191,23619
Polymers184,8652
Non-polymers6,37217
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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Gamma-aminobutyric acid type B receptor subunit ... , 2 types, 2 molecules AB

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


Mass: 91585.867 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): HEK293 GnTl- / 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: 93278.781 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): HEK293 GnTl- / Production host: Homo sapiens (human) / References: UniProt: O75899

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Sugars , 1 types, 4 molecules

#3: 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.: 4 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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

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Non-polymers , 4 types, 13 molecules

#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-U3G / (2R)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-{[(9Z)-octadec-9-enoyl]oxy}propyl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate


Mass: 766.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C43H76NO8P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-U3D / [(2R)-3-[(Z)-icos-11-enoyl]oxy-2-[(Z)-octadec-9-enoyl]oxypropyl] 2-(trimethylazaniumyl)ethyl phosphate / [(2~{R})-2-[(~{Z})-octadec-9-enoyl]oxy-3-[oxidanyl-[2-(trimethyl-$l^{4}-azanyl)ethoxy]phosphoryl]oxy-propyl] (~{Z})-icos-11-enoate


Mass: 814.167 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C46H88NO8P / 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

ComponentName: Heterodimer human GABA(B) receptor composed of GABA(B1b) and GABA(B2) subunits.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.193 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293 GnTI- / Plasmid: modified bacMam
Buffer solutionpH: 7.5
Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaClSodium chloride1
30.002 %Lauryl Maltose Neopentyl GlycolC47H88O221
40.0004 %Cholesteryl HemisuccinateC31H50O41
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: -2000 nm / Nominal defocus min: -500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K
Image recordingAverage exposure time: 12 sec. / Electron dose: 85 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3435
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2Leginonimage acquisition
4Gctf1.06CTF correction
7Coot0.8.9.0model fitting
9PHENIX1.14model refinement
10cryoSPARC2initial Euler assignmentNon-uniform refinement
11cryoSPARC2final Euler assignment
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1048241
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233737 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
14MQE

4mqe

A148-459
24MQE

4mqe

B153-466
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 68.95 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004611487
ELECTRON MICROSCOPYf_angle_d0.696915616
ELECTRON MICROSCOPYf_chiral_restr0.04381792
ELECTRON MICROSCOPYf_plane_restr0.00451888
ELECTRON MICROSCOPYf_dihedral_angle_d12.56166736

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