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- PDB-6wha: HTR2A bound to 25-CN-NBOH in complex with a mini-Galpha-q protein... -

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Entry
Database: PDB / ID: 6wha
TitleHTR2A bound to 25-CN-NBOH in complex with a mini-Galpha-q protein, beta/gamma subunits and an active-state stabilizing single-chain variable fragment (scFv16) obtained by cryo-electron microscopy (cryoEM)
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • G subunit q (Gi2-mini-Gq chimeric)
  • Soluble cytochrome b562,5-hydroxytryptamine receptor 2A chimera
  • single Fab chain (svFv16)
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / 5HT2A / G protein / 25-CN-NBOH / GPCR / cryo-EM / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of heat generation / protein localization to cytoskeleton / 1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine binding / Gq/11-coupled serotonin receptor activity / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / G protein-coupled serotonin receptor complex / neurofilament / Serotonin receptors / serotonin receptor activity ...positive regulation of heat generation / protein localization to cytoskeleton / 1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine binding / Gq/11-coupled serotonin receptor activity / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / G protein-coupled serotonin receptor complex / neurofilament / Serotonin receptors / serotonin receptor activity / cell body fiber / G protein-coupled serotonin receptor activity / phospholipase C-activating serotonin receptor signaling pathway / artery smooth muscle contraction / positive regulation of cytokine production involved in immune response / serotonin receptor signaling pathway / sensitization / urinary bladder smooth muscle contraction / neurotransmitter receptor activity / serotonin binding / negative regulation of synaptic transmission, glutamatergic / positive regulation of platelet aggregation / positive regulation of DNA biosynthetic process / temperature homeostasis / regulation of dopamine secretion / behavioral response to cocaine / negative regulation of potassium ion transport / detection of temperature stimulus involved in sensory perception of pain / protein tyrosine kinase activator activity / positive regulation of execution phase of apoptosis / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / positive regulation of fat cell differentiation / detection of mechanical stimulus involved in sensory perception of pain / positive regulation of vasoconstriction / release of sequestered calcium ion into cytosol / presynaptic modulation of chemical synaptic transmission / dendritic shaft / positive regulation of glycolytic process / glycolytic process / electron transport chain / caveola / memory / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / intracellular calcium ion homeostasis / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / positive regulation of inflammatory response / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / positive regulation of neuron apoptotic process / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / presynaptic membrane / virus receptor activity / GTPase binding / positive regulation of cytosolic calcium ion concentration / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / cytoplasmic vesicle / fibroblast proliferation / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / chemical synaptic transmission / G alpha (q) signalling events / postsynaptic membrane / Ras protein signal transduction / periplasmic space
Similarity search - Function
5-Hydroxytryptamine 2A receptor / 5-hydroxytryptamine receptor family / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily ...5-Hydroxytryptamine 2A receptor / 5-hydroxytryptamine receptor family / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / P-loop containing nucleotide triphosphate hydrolases / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-U0G / Soluble cytochrome b562 / 5-hydroxytryptamine receptor 2A / 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
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsSkiniotis, G. / Roth, B. / Kim, K. / Panova, O.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)R37 DA045657 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)RO1 MH112205 United States
CitationJournal: Cell / Year: 2020
Title: Structure of a Hallucinogen-Activated Gq-Coupled 5-HT Serotonin Receptor.
Authors: Kuglae Kim / Tao Che / Ouliana Panova / Jeffrey F DiBerto / Jiankun Lyu / Brian E Krumm / Daniel Wacker / Michael J Robertson / Alpay B Seven / David E Nichols / Brian K Shoichet / Georgios ...Authors: Kuglae Kim / Tao Che / Ouliana Panova / Jeffrey F DiBerto / Jiankun Lyu / Brian E Krumm / Daniel Wacker / Michael J Robertson / Alpay B Seven / David E Nichols / Brian K Shoichet / Georgios Skiniotis / Bryan L Roth /
Abstract: Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many ...Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
History
DepositionApr 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Soluble cytochrome b562,5-hydroxytryptamine receptor 2A chimera
B: G subunit q (Gi2-mini-Gq chimeric)
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: single Fab chain (svFv16)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)160,5136
Polymers160,2005
Non-polymers3121
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Mix purified HTR2A and G complex and scFv16, and then allow gel filtration.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9630 Å2
ΔGint-57 kcal/mol
Surface area49450 Å2

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Soluble cytochrome b562,5-hydroxytryptamine receptor 2A chimera / Cytochrome b-562 / 5-HT-2A / Serotonin receptor 2A


Mass: 56167.309 Da / Num. of mol.: 1
Fragment: cybC (UNP residues 23-127) + linker + HTR2A (UNP residues 66-404)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, HTR2A, HTR2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0ABE7, UniProt: P28223
#2: Protein G subunit q (Gi2-mini-Gq chimeric)


Mass: 28084.832 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)

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules CD

#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 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
#4: 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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Antibody / Non-polymers , 2 types, 2 molecules E

#5: Antibody single Fab chain (svFv16)


Mass: 28668.922 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-U0G / 4-(2-{[(2-hydroxyphenyl)methyl]amino}ethyl)-2,5-dimethoxybenzonitrile


Mass: 312.363 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H20N2O3 / Feature type: SUBJECT OF INVESTIGATION / Comment: agonist*YM

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Details

Has ligand of interestY
Has protein modificationY

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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: HTR2A bound to 25-CN-NBOH in complex with a mini-Galpha-q protein, beta/gamma subunits and an active-state stabilizing single-chain variable fragment (scFv16) obtained by cryo-electron microscopy (cryoEM)
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2100 mMNaCl1
30.001 %LMNG1
40.0001 %CHS1
50.00025 %GDN1
6100 uMTCEP1
71 mMMgCl21
810 uMGDP1
SpecimenConc.: 16.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / 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 FIELD / Calibrated magnification: 57050 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2500 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
Image recordingAverage exposure time: 4 sec. / Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2368

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2712613
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166497 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.95 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.018501
ELECTRON MICROSCOPYf_angle_d0.95711575
ELECTRON MICROSCOPYf_dihedral_angle_d17.7461204
ELECTRON MICROSCOPYf_chiral_restr0.0591347
ELECTRON MICROSCOPYf_plane_restr0.0061478

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