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- PDB-6kpf: Cryo-EM structure of a class A GPCR with G protein complex -

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Basic information

Entry
Database: PDB / ID: 6kpf
TitleCryo-EM structure of a class A GPCR with G protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 2Cannabinoid receptor type 2
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / Gi Protein
Function / homology
Function and homology information


cannabinoid receptor activity / negative regulation of mast cell activation / negative regulation of nitric-oxide synthase activity / negative regulation of synaptic transmission, GABAergic / negative regulation of action potential / leukocyte chemotaxis / G protein-coupled serotonin receptor binding / cardiac muscle cell apoptotic process / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling ...cannabinoid receptor activity / negative regulation of mast cell activation / negative regulation of nitric-oxide synthase activity / negative regulation of synaptic transmission, GABAergic / negative regulation of action potential / leukocyte chemotaxis / G protein-coupled serotonin receptor binding / cardiac muscle cell apoptotic process / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / sensory perception of taste / G-protein beta/gamma-subunit complex / cellular response to catecholamine stimulus / G protein-coupled acetylcholine receptor signaling pathway / alkylglycerophosphoethanolamine phosphodiesterase activity / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / cell cortex region / rhodopsin mediated signaling pathway / adenylate cyclase-activating dopamine receptor signaling pathway / photoreceptor outer segment membrane / G-protein beta-subunit binding / photoreceptor disc membrane / regulation of mitotic spindle organization / spectrin binding / negative regulation of synaptic transmission / G-protein beta/gamma-subunit complex binding / cellular response to forskolin / heterotrimeric G-protein complex / GTPase activating protein binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / extracellular vesicle / Wnt signaling pathway, calcium modulating pathway / cellular response to prostaglandin E stimulus / response to amphetamine / retina development in camera-type eye / phospholipase C-activating G protein-coupled receptor signaling pathway / GTPase binding / response to peptide hormone / sensory perception of pain / photoreceptor inner segment / G protein-coupled receptor binding / negative regulation of inflammatory response / extrinsic component of cytoplasmic side of plasma membrane / GDP binding / cell body / midbody / lysosomal membrane / cellular response to hypoxia / positive regulation of cytosolic calcium ion concentration / platelet activation / Ras protein signal transduction / cell population proliferation / protein folding / perikaryon / response to lipopolysaccharide / immune response / cell cycle / GTPase activity / centrosome / cell division / inflammatory response / synapse / protein-containing complex binding / G protein-coupled receptor signaling pathway / membrane raft / GTP binding / dendrite / signal transduction / integral component of plasma membrane / magnesium ion binding / cell / extracellular exosome / membrane / plasma membrane / nucleus / cytosol / cytoplasm
WD40/YVTN repeat-like-containing domain superfamily / G-protein, beta subunit / GPCR, rhodopsin-like, 7TM / G protein-coupled receptor, rhodopsin-like / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / WD40 repeat, conserved site / Cannabinoid receptor type 2 / G-protein beta WD-40 repeat / WD40 repeat ...WD40/YVTN repeat-like-containing domain superfamily / G-protein, beta subunit / GPCR, rhodopsin-like, 7TM / G protein-coupled receptor, rhodopsin-like / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / WD40 repeat, conserved site / Cannabinoid receptor type 2 / G-protein beta WD-40 repeat / WD40 repeat / WD40-repeat-containing domain / P-loop containing nucleoside triphosphate hydrolase / G-protein gamma-like domain superfamily / WD40-repeat-containing domain superfamily / G-protein, gamma subunit / Cannabinoid receptor family / G protein alpha subunit, helical insertion / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit
Cannabinoid receptor 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
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLi, X.T. / Hua, T. / Wu, L.J. / Makriyannis, A. / Shen, L. / Wang, Y.X. / 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 / 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

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Structure visualization

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Structure viewerMolecule:
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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 2
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,6466
Polymers153,2015
Non-polymers4461
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11400 Å2
ΔGint-71 kcal/mol
Surface area46660 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: 40415.031 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: 37416.930 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 2 / Cannabinoid receptor type 2 / hCB2 / CX5


Mass: 39722.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CNR2, CB2A, CB2B / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P34972
#5: Antibody scFv16


Mass: 27784.896 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: GPCR-G protein complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Molecular weightValue: 130 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

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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: 4496
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
1RELION3.03particle selection
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: 5262021
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 960302 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 45.8 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC_mask
Atomic model buildingPDB-ID: 6N4B
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.0078978
f_angle_d0.87112175
f_dihedral_angle_d11.7055299
f_chiral_restr0.0531392
f_plane_restr0.0071528

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