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- EMDB-0745: Cryo-EM structure of CB1-G protein complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0745
TitleCryo-EM structure of CB1-G protein complex
Map data
SampleCB1-G protein complex
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 1Cannabinoid receptor type 1
  • scFv16
  • ligand
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 / photoreceptor outer segment membrane / adenylate cyclase-activating dopamine receptor signaling pathway / 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 / extracellular vesicle / G protein-coupled receptor activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Wnt signaling pathway, calcium modulating 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 / positive regulation of cytosolic calcium ion concentration / growth cone / 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 / synapse / G protein-coupled receptor signaling pathway / 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
G protein alpha subunit, helical insertion / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / G-protein beta WD-40 repeat / G protein-coupled receptor, rhodopsin-like / P-loop containing nucleoside triphosphate hydrolase / Cannabinoid receptor type 1 ...G protein alpha subunit, helical insertion / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / G-protein beta WD-40 repeat / G protein-coupled receptor, rhodopsin-like / P-loop containing nucleoside triphosphate hydrolase / Cannabinoid receptor type 1 / WD40 repeat, conserved site / G-protein alpha subunit, group I / WD40 repeat / Cannabinoid receptor family / G-protein gamma-like domain superfamily / G-protein, gamma subunit / GPCR, rhodopsin-like, 7TM / WD40-repeat-containing domain superfamily / G-protein, beta subunit
Cannabinoid receptor 1 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsHua T / Li XT / Wu LJ / Makriyannis A / Wang YX / Shen L / Liu ZJ
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 ReportPDB-ID: 6kpg

SummaryFull reportAbout validation report
History
DepositionAug 15, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateMar 11, 2020-
Current statusMar 11, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kpg
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0745.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 224 pix.
= 232.96 Å
1.04 Å/pix.
x 224 pix.
= 232.96 Å
1.04 Å/pix.
x 224 pix.
= 232.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.11882624 - 0.18688558
Average (Standard dev.)0.0000116533 (±0.0043318244)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 232.95999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z232.960232.960232.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.1190.1870.000

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Supplemental data

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Sample components

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Entire CB1-G protein complex

EntireName: CB1-G protein complex / Number of components: 7

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Component #1: protein, CB1-G protein complex

ProteinName: CB1-G protein complex / Recombinant expression: No
MassExperimental: 140 MDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Guanine nucleotide-binding protein G(i) subunit alpha-1

ProteinName: Guanine nucleotide-binding protein G(i) subunit alpha-1
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 40.283836 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subuni...

ProteinName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 38.045629 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #4: protein, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subuni...

ProteinName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 7.861143 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, Cannabinoid receptor 1

ProteinName: Cannabinoid receptor 1Cannabinoid receptor type 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 52.843691 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Mammalian expression vector HA-MCS-pcDNA3.1 (others)

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Component #6: protein, scFv16

ProteinName: scFv16 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.742861 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #7: ligand, (6~{a}~{R},9~{R},10~{a}~{R})-9-(hydroxymethyl)-3-(8-isoth...

LigandName: (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
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.445658 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 7.5
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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.333 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1000.0 - -2000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (70.0 - 70.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5577

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 505644
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 6N4B
Overall bvalue: 72.4
Output model

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