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- PDB-7t32: CryoEM structure of the adenosine 2A receptor-BRIL/Anti BRIL Fab ... -

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Basic information

Entry
Database: PDB / ID: 7t32
TitleCryoEM structure of the adenosine 2A receptor-BRIL/Anti BRIL Fab complex with ZM241385
ComponentsAdenosine receptor A2a/Soluble cytochrome b562 Fusion Protein
KeywordsMEMBRANE PROTEIN / A2AAR / GPCR / ADENOSINE RECEPTOR
Function / homology
Function and homology information


positive regulation of acetylcholine secretion, neurotransmission / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / sensory perception ...positive regulation of acetylcholine secretion, neurotransmission / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / sensory perception / Surfactant metabolism / positive regulation of urine volume / synaptic transmission, dopaminergic / type 5 metabotropic glutamate receptor binding / negative regulation of vascular permeability / positive regulation of glutamate secretion / synaptic transmission, cholinergic / intermediate filament / presynaptic active zone / response to caffeine / blood circulation / eating behavior / inhibitory postsynaptic potential / alpha-actinin binding / regulation of calcium ion transport / neuron projection morphogenesis / asymmetric synapse / membrane depolarization / axolemma / phagocytosis / positive regulation of synaptic transmission, glutamatergic / prepulse inhibition / cellular defense response / regulation of mitochondrial membrane potential / presynaptic modulation of chemical synaptic transmission / astrocyte activation / response to amphetamine / central nervous system development / positive regulation of apoptotic signaling pathway / positive regulation of long-term synaptic potentiation / excitatory postsynaptic potential / positive regulation of protein secretion / positive regulation of synaptic transmission, GABAergic / synaptic transmission, glutamatergic / locomotory behavior / apoptotic signaling pathway / electron transport chain / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / negative regulation of inflammatory response / vasodilation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / blood coagulation / cell-cell signaling / presynaptic membrane / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / negative regulation of neuron apoptotic process / postsynaptic membrane / periplasmic space / calmodulin binding / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / inflammatory response / iron ion binding / response to xenobiotic stimulus / negative regulation of cell population proliferation / neuronal cell body / apoptotic process / heme binding / lipid binding / dendrite / regulation of DNA-templated transcription / protein-containing complex binding / glutamatergic synapse / enzyme binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Adenosine A2A receptor / Adenosine receptor / Cytochrome c/b562 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / Four Helix Bundle (Hemerythrin (Met), subunit A) / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like ...Adenosine A2A receptor / Adenosine receptor / Cytochrome c/b562 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / Four Helix Bundle (Hemerythrin (Met), subunit A) / 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) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-ZMA / Soluble cytochrome b562 / Adenosine receptor A2a
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhang, K.H. / Wu, H. / Hoppe, N. / Manglik, A. / Cheng, Y.F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD) United States
Citation
Journal: Nat Commun / Year: 2022
Title: Fusion protein strategies for cryo-EM study of G protein-coupled receptors.
Authors: Kaihua Zhang / Hao Wu / Nicholas Hoppe / Aashish Manglik / Yifan Cheng /
Abstract: Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, ...Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although a similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which lead to structures of antagonist bound A adenosine receptor at 3.4 Å resolution and unliganded Smoothened at 3.7 Å resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.
#1: Journal: Science / Year: 2012
Title: Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions
Authors: Liu, W.
History
DepositionDec 6, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,7532
Polymers43,4161
Non-polymers3371
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein / Cytochrome b-562


Mass: 43415.855 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: ADORA2A, ADORA2, cybC / Production host: Homo sapiens (human) / References: UniProt: P29274, UniProt: P0ABE7
#2: Chemical ChemComp-ZMA / 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol


Mass: 337.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H15N7O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: antagonist*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: A2A adenosine receptor-BRIL/Anti BRIL Fab complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: OTHER

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 67 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215946 / Symmetry type: POINT

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