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- PDB-6kpg: Cryo-EM structure of CB1-G protein complex -

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Basic information

Entry
Database: PDB / ID: 6kpg
TitleCryo-EM structure of CB1-G protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 1Cannabinoid receptor type 1
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / G protein / cryo-EM
Function / homology
Function and homology information


regulation of penile erection / cannabinoid receptor activity / cannabinoid signaling pathway / negative regulation of dopamine secretion / negative regulation of mast cell activation / regulation of feeding behavior / regulation of metabolic process / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / negative regulation of nitric-oxide synthase activity / negative regulation of fatty acid beta-oxidation ...regulation of penile erection / cannabinoid receptor activity / cannabinoid signaling pathway / negative regulation of dopamine secretion / negative regulation of mast cell activation / regulation of feeding behavior / regulation of metabolic process / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / negative regulation of nitric-oxide synthase activity / negative regulation of fatty acid beta-oxidation / positive regulation of acute inflammatory response to antigenic stimulus / negative regulation of serotonin secretion / negative regulation of action potential / G protein-coupled serotonin receptor binding / positive regulation of protein localization to cell cortex / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / cell cortex region / positive regulation of blood pressure / positive regulation of fever generation / cardiac muscle cell apoptotic process / sensory perception of taste / G protein-coupled acetylcholine receptor signaling pathway / G-protein beta/gamma-subunit complex / cellular response to catecholamine stimulus / G-protein gamma-subunit binding / alkylglycerophosphoethanolamine phosphodiesterase activity / rhodopsin mediated signaling pathway / axonal fasciculation / regulation of cAMP-mediated signaling / regulation of mitotic spindle organization / regulation of synaptic transmission, GABAergic / adenylate cyclase-activating dopamine receptor signaling pathway / photoreceptor outer segment membrane / cellular response to forskolin / negative regulation of synaptic transmission / G-protein beta-subunit binding / photoreceptor disc membrane / spectrin binding / G-protein beta/gamma-subunit complex binding / heterotrimeric G-protein complex / GTPase activating protein binding / maternal process involved in female pregnancy / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / response to morphine / G protein-coupled receptor activity / extracellular vesicle / Wnt signaling pathway, calcium modulating pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / cellular response to prostaglandin E stimulus / regulation of synaptic transmission, glutamatergic / phospholipase C-activating G protein-coupled receptor signaling pathway / response to cocaine / adenylate cyclase-activating G protein-coupled receptor signaling pathway / negative regulation of blood pressure / GTPase binding / response to peptide hormone / regulation of insulin secretion / G protein-coupled receptor binding / sensory perception of pain / retina development in camera-type eye / photoreceptor inner segment / response to nicotine / positive regulation of neuron projection development / response to nutrient / memory / presynaptic membrane / GDP binding / cell population proliferation / cell body / midbody / lysosomal membrane / drug binding / actin cytoskeleton / cellular response to hypoxia / growth cone / positive regulation of cytosolic calcium ion concentration / spermatogenesis / glucose homeostasis / Ras protein signal transduction / platelet activation / mitochondrial outer membrane / protein folding / response to lipopolysaccharide / response to ethanol / aging / cell cycle / GTPase activity / cell division / centrosome / G protein-coupled receptor signaling pathway / synapse / membrane raft / positive regulation of apoptotic process / GTP binding / dendrite / protein-containing complex binding / magnesium ion binding / signal transduction / integral component of plasma membrane / extracellular exosome
WD domain, G-beta repeat / G-protein alpha subunit / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / Cannabinoid receptor type 1 / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / G-protein, beta subunit / WD40 repeat / G-protein, gamma subunit ...WD domain, G-beta repeat / G-protein alpha subunit / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / Cannabinoid receptor type 1 / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / G-protein, beta subunit / WD40 repeat / G-protein, gamma subunit / Cannabinoid receptor family / G protein alpha subunit, helical insertion / G-protein gamma-like domain / WD40/YVTN repeat-like-containing domain superfamily / Guanine nucleotide-binding protein, beta subunit / GPCR, rhodopsin-like, 7TM / WD40-repeat-containing domain / WD40 repeat, conserved site / G-protein beta WD-40 repeat / P-loop containing nucleoside triphosphate hydrolase / G-protein gamma-like domain superfamily / WD40-repeat-containing domain superfamily / GGL domain / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / YVTN repeat-like/Quinoprotein amine dehydrogenase / Methylamine Dehydrogenase; Chain H / 7 Propeller / Few Secondary Structures / Irregular / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Guanine nucleotide-binding protein G(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Cannabinoid receptor 1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsHua, T. / Li, X.T. / Wu, L.J. / Makriyannis, A. / Wang, Y.X. / Shen, L. / Liu, Z.J.
CitationJournal: Cell / Year: 2020
Title: Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-G Complex Structures.
Authors: Tian Hua / Xiaoting Li / Lijie Wu / Christos Iliopoulos-Tsoutsouvas / Yuxia Wang / Meng Wu / Ling Shen / Christina A Johnston / Spyros P Nikas / Feng Song / Xiyong Song / Shuguang Yuan / ...Authors: Tian Hua / Xiaoting Li / Lijie Wu / Christos Iliopoulos-Tsoutsouvas / Yuxia Wang / Meng Wu / Ling Shen / Christina A Johnston / Spyros P Nikas / Feng Song / Xiyong Song / Shuguang Yuan / Qianqian Sun / Yiran Wu / Shan Jiang / Travis W Grim / Othman Benchama / Edward L Stahl / Nikolai Zvonok / Suwen Zhao / Laura M Bohn / Alexandros Makriyannis / Zhi-Jie Liu /
Abstract: Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, ...Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 15, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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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
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
R: Cannabinoid receptor 1
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,2236
Polymers166,7775
Non-polymers4461
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11170 Å2
ΔGint-71 kcal/mol
Surface area46840 Å2

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Components

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

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


Mass: 40283.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38045.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / 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 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein / Antibody / Non-polymers , 3 types, 3 molecules RS

#4: Protein Cannabinoid receptor 1 / Cannabinoid receptor type 1 / CB1 / CANN6


Mass: 52843.691 Da / Num. of mol.: 1 / Mutation: E273K, T283V, R340E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CNR1, CNR
Production host: Mammalian expression vector HA-MCS-pcDNA3.1 (others)
References: UniProt: P21554
#5: Antibody scFv16


Mass: 27742.861 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Chemical ChemComp-8D0 / (6~{a}~{R},9~{R},10~{a}~{R})-9-(hydroxymethyl)-3-(8-isothiocyanato-2-methyl-octan-2-yl)-6,6-dimethyl-6~{a},7,8,9,10,10~{a}-hexahydrobenzo[c]chromen-1-ol


Mass: 445.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H39NO3S / 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: CB1-G protein complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 140 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 1 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 %

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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 / Nominal defocus max: -2000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 1.333 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5577
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 45 / Used frames/image: 1-45

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.7.3image acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9RELION3.03initial Euler assignment
10RELION3.03final Euler assignment
11RELION3.03classification
12RELION3.033D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2186340
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 505644 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 72.4 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6N4B
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0138988
ELECTRON MICROSCOPYf_angle_d1.04912177
ELECTRON MICROSCOPYf_dihedral_angle_d11.4755319
ELECTRON MICROSCOPYf_chiral_restr0.0631399
ELECTRON MICROSCOPYf_plane_restr0.0071524

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