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- EMDB-25648: CryoEM structure of the adenosine 2A receptor-BRIL/Anti BRIL Fab ... -

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Basic information

Entry
Database: EMDB / ID: EMD-25648
TitleCryoEM structure of the adenosine 2A receptor-BRIL/Anti BRIL Fab complex with ZM241385
Map dataStructure of the adenosine 2A receptor-BRIL/Anti BRIL Fab with ZM241385
Sample
  • Complex: A2A adenosine receptor-BRIL/Anti BRIL Fab complex
    • Protein or peptide: Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein
  • Ligand: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol
KeywordsA2AAR / GPCR / ADENOSINE RECEPTOR / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of norepinephrine secretion / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / positive regulation of acetylcholine secretion, neurotransmission / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism / type 5 metabotropic glutamate receptor binding ...regulation of norepinephrine secretion / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / positive regulation of acetylcholine secretion, neurotransmission / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism / type 5 metabotropic glutamate receptor binding / negative regulation of vascular permeability / positive regulation of urine volume / intermediate filament / response to caffeine / blood circulation / presynaptic active zone / sensory perception / eating behavior / positive regulation of glutamate secretion / synaptic transmission, cholinergic / regulation of calcium ion transport / alpha-actinin binding / synaptic transmission, dopaminergic / membrane depolarization / asymmetric synapse / axolemma / prepulse inhibition / cellular defense response / phagocytosis / neuron projection morphogenesis / positive regulation of synaptic transmission, glutamatergic / presynaptic modulation of chemical synaptic transmission / astrocyte activation / regulation of mitochondrial membrane potential / excitatory postsynaptic potential / positive regulation of protein secretion / locomotory behavior / central nervous system development / positive regulation of long-term synaptic potentiation / electron transport chain / synaptic transmission, glutamatergic / vasodilation / blood coagulation / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / cell-cell signaling / adenylate cyclase-activating G protein-coupled receptor signaling pathway / presynaptic membrane / negative regulation of neuron apoptotic process / G alpha (s) signalling events / positive regulation of ERK1 and ERK2 cascade / calmodulin binding / electron transfer activity / periplasmic space / postsynaptic membrane / response to xenobiotic stimulus / inflammatory response / iron ion binding / negative regulation of cell population proliferation / neuronal cell body / heme binding / apoptotic process / regulation of DNA-templated transcription / lipid binding / dendrite / protein-containing complex binding / glutamatergic synapse / enzyme binding / membrane / identical protein binding / plasma membrane
Similarity search - Function
Adenosine A2A receptor / Adenosine receptor / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile.
Similarity search - Domain/homology
Soluble cytochrome b562 / Adenosine receptor A2a
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhang KH / Wu H / Hoppe N / Manglik A / Cheng YF
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25648.png

Downloads & links

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Map

FileDownload / File: emd_25648.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the adenosine 2A receptor-BRIL/Anti BRIL Fab with ZM241385
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.9305815 - 3.3283527
Average (Standard dev.)0.0019232043 (±0.047693416)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : A2A adenosine receptor-BRIL/Anti BRIL Fab complex

EntireName: A2A adenosine receptor-BRIL/Anti BRIL Fab complex
Components
  • Complex: A2A adenosine receptor-BRIL/Anti BRIL Fab complex
    • Protein or peptide: Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein
  • Ligand: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol

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Supramolecule #1: A2A adenosine receptor-BRIL/Anti BRIL Fab complex

SupramoleculeName: A2A adenosine receptor-BRIL/Anti BRIL Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein

MacromoleculeName: Adenosine receptor A2a/Soluble cytochrome b562 Fusion Protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.415855 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VYITVELAIA VLAILGNVLV CWAVWLNSNL QNVTNYFVVS LAAADIAVGV LAIPFAITIS TGFCAACHGC LFIACFVLVL TQSSIFSLL AIAIDRYIAI RIPLRYNGLV TGTRAKGIIA ICWVLSFAIG LTPMLGWNNC GQPKEGKNHS QGCGEGQVAC L FEDVVPMN ...String:
VYITVELAIA VLAILGNVLV CWAVWLNSNL QNVTNYFVVS LAAADIAVGV LAIPFAITIS TGFCAACHGC LFIACFVLVL TQSSIFSLL AIAIDRYIAI RIPLRYNGLV TGTRAKGIIA ICWVLSFAIG LTPMLGWNNC GQPKEGKNHS QGCGEGQVAC L FEDVVPMN YMVYFNFFAC VLVPLLLMLG VYLRIFLAAR RQLADLEDNW ETLNDNLKVI EKADNAAQVK DALTKMRAAA LD AQKATPP KLEDKSPDSP EMKDFRHGFD ILVGQIDDAL KLANEGKVKE AQAAAEQLKT TRNAYIQKYL ERARSTLQKE VHA AKSLAI IVGLFALCWL PLHIINCFTF FCPDCSHAPL WLMYLAIVLS HTNSVVNPFI YAYRIREFRQ TFRKI

UniProtKB: Adenosine receptor A2a, Soluble cytochrome b562, Adenosine receptor A2a

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Macromolecule #2: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5...

MacromoleculeName: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol
type: ligand / ID: 2 / Number of copies: 1 / Formula: ZMA
Molecular weightTheoretical: 337.336 Da
Chemical component information

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 / antagonist*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 67.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 215946
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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