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- EMDB-0744: Cryo-EM structure of a class A GPCR with G protein complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0744
TitleCryo-EM structure of a class A GPCR with G protein complex
Map data
SampleGPCR-G protein complex
  • (Guanine nucleotide-binding protein ...) x 3
  • Cannabinoid receptor 2Cannabinoid receptor type 2
  • scFv16
  • ligand
Function / homology
Function and homology information


cannabinoid receptor activity / cannabinoid signaling pathway / negative regulation of mast cell activation / regulation of metabolic process / 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 / positive regulation of protein localization to cell cortex ...cannabinoid receptor activity / cannabinoid signaling pathway / negative regulation of mast cell activation / regulation of metabolic process / 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 / positive regulation of protein localization to cell cortex / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / cardiac muscle cell apoptotic process / sensory perception of taste / regulation of cAMP-mediated signaling / cell cortex region / G-protein beta/gamma-subunit complex / cellular response to catecholamine stimulus / G-protein gamma-subunit binding / G protein-coupled acetylcholine receptor signaling pathway / alkylglycerophosphoethanolamine phosphodiesterase activity / rhodopsin mediated signaling pathway / regulation of mitotic spindle organization / adenylate cyclase-activating dopamine receptor signaling pathway / photoreceptor outer segment membrane / G-protein beta-subunit binding / cellular response to forskolin / negative regulation of synaptic transmission / photoreceptor disc membrane / spectrin binding / G-protein beta/gamma-subunit complex binding / heterotrimeric G-protein complex / GTPase activating protein binding / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Wnt signaling pathway, calcium modulating pathway / G protein-coupled receptor activity / extracellular vesicle / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / cellular response to prostaglandin E stimulus / response to amphetamine / phospholipase C-activating G protein-coupled receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / GTPase binding / response to peptide hormone / retina development in camera-type eye / G protein-coupled receptor binding / sensory perception of pain / photoreceptor inner segment / extrinsic component of cytoplasmic side of plasma membrane / GDP binding / negative regulation of inflammatory response / cell population proliferation / cell body / midbody / lysosomal membrane / cellular response to hypoxia / positive regulation of cytosolic calcium ion concentration / platelet activation / Ras protein signal transduction / protein folding / perikaryon / response to lipopolysaccharide / immune response / cell cycle / inflammatory response / GTPase activity / cell division / centrosome / G protein-coupled receptor signaling pathway / synapse / membrane raft / GTP binding / dendrite / protein-containing complex binding / signal transduction / endoplasmic reticulum / magnesium ion binding / integral component of plasma membrane / extracellular exosome / membrane / plasma membrane / nucleus / cytosol / cytoplasm
WD40/YVTN repeat-like-containing domain superfamily / Cannabinoid receptor type 2 / Guanine nucleotide-binding protein, beta subunit / WD40-repeat-containing domain / G protein-coupled receptor, rhodopsin-like / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Guanine nucleotide binding protein (G-protein), alpha subunit / P-loop containing nucleoside triphosphate hydrolase / G-protein alpha subunit, group I ...WD40/YVTN repeat-like-containing domain superfamily / Cannabinoid receptor type 2 / Guanine nucleotide-binding protein, beta subunit / WD40-repeat-containing domain / G protein-coupled receptor, rhodopsin-like / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Guanine nucleotide binding protein (G-protein), alpha subunit / P-loop containing nucleoside triphosphate hydrolase / G-protein alpha subunit, group I / GPCR, rhodopsin-like, 7TM / G-protein, beta subunit / G-protein gamma-like domain superfamily / WD40-repeat-containing domain superfamily / WD40 repeat / G-protein, gamma subunit / Cannabinoid receptor family / G protein alpha subunit, helical insertion / G-protein gamma-like domain
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLi XT / Hua T / Wu LJ / Makriyannis A / Shen L / Wang YX / 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: 6kpf

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-6kpf
  • 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_0744.map.gz / Format: CCP4 / Size: 64 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 256 pix.
= 266.24 Å
1.04 Å/pix.
x 256 pix.
= 266.24 Å
1.04 Å/pix.
x 256 pix.
= 266.24 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-0.14660877 - 0.22728585
Average (Standard dev.)0.00001193136 (±0.0048254933)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1470.2270.000

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Supplemental data

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Sample components

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Entire GPCR-G protein complex

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

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Component #1: protein, GPCR-G protein complex

ProteinName: GPCR-G protein complex / Recombinant expression: No
MassExperimental: 130 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: 40.415031 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: 37.41693 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 2

ProteinName: Cannabinoid receptor 2Cannabinoid receptor type 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 39.722715 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: protein, scFv16

ProteinName: scFv16 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.784896 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

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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: 4496

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 960302
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: CC_mask / Refinement space: REAL
Input PDB model: 6N4B
Overall bvalue: 45.8
Output model

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