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- EMDB-24551: MicroED structure of the human adenosine receptor at 2.8A -

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Basic information

Entry
Database: EMDB / ID: EMD-24551
TitleMicroED structure of the human adenosine receptor at 2.8A
Map data2mFo-dFc map of A2A AR by MicroED
Sample
  • Complex: Adenosine receptor A2a/Soluble cytochrome b562 chimera
    • Protein or peptide: Adenosine receptor A2a/Soluble cytochrome b562 chimera
  • 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
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM ION
  • Ligand: water
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / G protein-coupled adenosine receptor signaling pathway / response to purine-containing compound / sensory perception / positive regulation of urine volume ...positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / G protein-coupled adenosine receptor signaling pathway / response to purine-containing compound / sensory perception / positive regulation of urine volume / NGF-independant TRKA activation / Surfactant metabolism / synaptic transmission, dopaminergic / : / inhibitory postsynaptic potential / negative regulation of vascular permeability / synaptic transmission, cholinergic / type 5 metabotropic glutamate receptor binding / positive regulation of glutamate secretion / blood circulation / response to caffeine / intermediate filament / eating behavior / presynaptic active zone / alpha-actinin binding / membrane depolarization / regulation of calcium ion transport / asymmetric synapse / axolemma / : / cellular defense response / prepulse inhibition / phagocytosis / response to amphetamine / presynaptic modulation of chemical synaptic transmission / excitatory postsynaptic potential / positive regulation of synaptic transmission, glutamatergic / neuron projection morphogenesis / regulation of mitochondrial membrane potential / synaptic transmission, glutamatergic / positive regulation of long-term synaptic potentiation / locomotory behavior / central nervous system development / astrocyte activation / positive regulation of synaptic transmission, GABAergic / positive regulation of protein secretion / apoptotic signaling pathway / electron transport chain / positive regulation of apoptotic signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / negative regulation of inflammatory response / vasodilation / blood coagulation / cell-cell signaling / presynaptic membrane / G alpha (s) signalling events / postsynaptic membrane / negative regulation of neuron apoptotic process / periplasmic space / electron transfer activity / calmodulin binding / response to xenobiotic stimulus / inflammatory response / iron ion binding / negative regulation of cell population proliferation / neuronal cell body / glutamatergic synapse / lipid binding / apoptotic process / dendrite / heme binding / protein-containing complex binding / regulation of DNA-templated transcription / enzyme binding / identical protein binding / membrane / 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. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Soluble cytochrome b562 / Adenosine receptor A2a
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodelectron crystallography / cryo EM / Resolution: 2.79 Å
AuthorsMartynowycz MW / Shiriaeva A
Funding support United States, 4 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127086 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124152 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP.
Authors: Michael W Martynowycz / Anna Shiriaeva / Xuanrui Ge / Johan Hattne / Brent L Nannenga / Vadim Cherezov / Tamir Gonen /
Abstract: G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining ...G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.
History
DepositionJul 26, 2021-
Header (metadata) releaseSep 8, 2021-
Map releaseSep 8, 2021-
UpdateOct 18, 2023-
Current statusOct 18, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.92597
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.92597
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rm5
  • Surface level: 0.92597
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rm5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24551.png

Downloads & links

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Map

FileDownload / File: emd_24551.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2mFo-dFc map of A2A AR by MicroED
Voxel sizeX: 0.667 Å / Y: 0.669 Å / Z: 0.698 Å
Density
Contour LevelBy AUTHOR: 0.92597 / Movie #1: 0.92597
Minimum - Maximum-4.4147315 - 5.765004
Average (Standard dev.)-0.005944546 (±0.9259675)
SymmetrySpace group: 20
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-113-15-40
Dimensions16274106
Spacing60270200
CellA: 40.02 Å / B: 180.63 Å / C: 139.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6670.6690.698
M x/y/z60270200
origin x/y/z0.0000.0000.000
length x/y/z40.020180.630139.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-113-15
NX/NY/NZ10616274
MAP C/R/S321
start NC/NR/NS-15-113-40
NC/NR/NS74162106
D min/max/mean-4.4155.765-0.006

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Supplemental data

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Sample components

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Entire : Adenosine receptor A2a/Soluble cytochrome b562 chimera

EntireName: Adenosine receptor A2a/Soluble cytochrome b562 chimera
Components
  • Complex: Adenosine receptor A2a/Soluble cytochrome b562 chimera
    • Protein or peptide: Adenosine receptor A2a/Soluble cytochrome b562 chimera
  • 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
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: Adenosine receptor A2a/Soluble cytochrome b562 chimera

SupramoleculeName: Adenosine receptor A2a/Soluble cytochrome b562 chimera
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.72 KDa

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Macromolecule #1: Adenosine receptor A2a/Soluble cytochrome b562 chimera

MacromoleculeName: Adenosine receptor A2a/Soluble cytochrome b562 chimera
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.974281 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDGAPPIMG SSVYITVELA IAVLAILGNV LVCWAVWLNS NLQNVTNYFV VSLAAADIAV GVLAIPFAI TISTGFCAAC HGCLFIACFV LVLTQSSIFS LLAIAIDRYI AIRIPLRYNG LVTGTRAKGI IAICWVLSFA I GLTPMLGW ...String:
MKTIIALSYI FCLVFADYKD DDDGAPPIMG SSVYITVELA IAVLAILGNV LVCWAVWLNS NLQNVTNYFV VSLAAADIAV GVLAIPFAI TISTGFCAAC HGCLFIACFV LVLTQSSIFS LLAIAIDRYI AIRIPLRYNG LVTGTRAKGI IAICWVLSFA I GLTPMLGW NNCGQPKEGK NHSQGCGEGQ VACLFEDVVP MNYMVYFNFF ACVLVPLLLM LGVYLRIFLA ARRQLADLED NW ETLNDNL KVIEKADNAA QVKDALTKMR AAALDAQKAT PPKLEDKSPD SPEMKDFRHG FDILVGQIDD ALKLANEGKV KEA QAAAEQ LKTTRNAYIQ KYLERARSTL QKEVHAAKSL AIIVGLFALC WLPLHIINCF TFFCPDCSHA PLWLMYLAIV LSHT NSVVN PFIYAYRIRE FRQTFRKIIR SHVLRQQEPF KAHHHHHHHH HH

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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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 4 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration10 mg/mL
BufferpH: 5
Details: 25-28% (v/v) PEG 400, 0.04-0.06M sodium thiocyanate, 2% (v/v) 2,5-hexanediol, 100mM sodium citrate, pH 5.0, Lipid Cubic Phase (LCP), temperature 293K
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 83.0 K
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number diffraction images: 134 / Average exposure time: 3.0 sec. / Average electron dose: 0.01 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 1900 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -40.0, 40.0, 0.6
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsCetaD binned by 2
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX
Molecular replacementSoftware - Name: PHASER
Symmetry determination software listSoftware - Name: DIALS
Crystallography statisticsNumber intensities measured: 37130 / Number structure factors: 10071 / Fourier space coverage: 77 / R merge: 0.299 / Overall phase error: 30 / Overall phase residual: 0 / Phase error rejection criteria: none / High resolution: 2.79 Å / Shell - Shell ID: 1 / Shell - High resolution: 37.91 Å / Shell - Low resolution: 2.79 Å / Shell - Number structure factors: 10071 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 77 / Shell - Multiplicity: 3.7

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Atomic model buiding 1

Initial modelPDB ID:

4eiy


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 43 / Target criteria: Maximum likelihood
Output model

PDB-7rm5:
MicroED structure of the human adenosine receptor at 2.8A

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