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- EMDB-29586: MicroED structure of A2A from plasma milled lamellae -

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Basic information

Entry
Database: EMDB / ID: EMD-29586
TitleMicroED structure of A2A from plasma milled lamellae
Map dataStructure of the human A2A adenosine receptor from plasma milled lamellae
Sample
  • Complex: A2A BRIL Adenosine receptor
    • Protein or peptide: Adenosine receptor A2a,Soluble cytochrome b562
  • 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: OLEIC ACID
  • Ligand: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
  • Ligand: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
  • Ligand: SODIUM IONSodium
  • Ligand: water
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 / protein kinase C-activating G protein-coupled receptor signaling pathway / synaptic transmission, dopaminergic / inhibitory postsynaptic potential / negative regulation of vascular permeability / type 5 metabotropic glutamate receptor binding / positive regulation of glutamate secretion / blood circulation / synaptic transmission, cholinergic / response to caffeine / intermediate filament / eating behavior / presynaptic active zone / alpha-actinin binding / membrane depolarization / regulation of calcium ion transport / asymmetric synapse / axolemma / response to inorganic substance / cellular defense response / prepulse inhibition / phagocytosis / positive regulation of apoptotic signaling pathway / response to amphetamine / excitatory postsynaptic potential / presynaptic modulation of chemical synaptic transmission / positive regulation of synaptic transmission, glutamatergic / neuron projection morphogenesis / regulation of mitochondrial membrane potential / locomotory behavior / synaptic transmission, glutamatergic / central nervous system development / positive regulation of long-term synaptic potentiation / astrocyte activation / positive regulation of synaptic transmission, GABAergic / apoptotic signaling pathway / positive regulation of protein secretion / 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 / electron transfer activity / periplasmic space / calmodulin binding / response to xenobiotic stimulus / inflammatory response / iron ion binding / negative regulation of cell population proliferation / neuronal cell body / dendrite / glutamatergic synapse / apoptotic process / lipid binding / heme binding / protein-containing complex binding / regulation of DNA-templated transcription / 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. / 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: 2.0 Å
AuthorsMartynowycz MW / Shiriaeva A / Clabbers MTB / Nicolas WJ / Weaver SJ / Hattne J / Gonen T
Funding support United States, 2 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
CitationJournal: Nat Commun / Year: 2023
Title: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals.
Authors: Michael W Martynowycz / Anna Shiriaeva / Max T B Clabbers / William J Nicolas / Sara J Weaver / Johan Hattne / Tamir Gonen /
Abstract: Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction ...Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
History
DepositionJan 26, 2023-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29586.png

Downloads & links

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Map

FileDownload / File: emd_29586.map.gz / Format: CCP4 / Size: 14.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the human A2A adenosine receptor from plasma milled lamellae
Voxel sizeX: 0.4885 Å / Y: 0.4885 Å / Z: 0.48365 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.0607924 - 7.3465424
Average (Standard dev.)-0.0035041242 (±0.9324466)
SymmetrySpace group: 20
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-154-25-58
Dimensions221118145
Spacing80360288
CellA: 39.08 Å / B: 175.86002 Å / C: 139.29 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : A2A BRIL Adenosine receptor

EntireName: A2A BRIL Adenosine receptor
Components
  • Complex: A2A BRIL Adenosine receptor
    • Protein or peptide: Adenosine receptor A2a,Soluble cytochrome b562
  • 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: OLEIC ACID
  • Ligand: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
  • Ligand: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: A2A BRIL Adenosine receptor

SupramoleculeName: A2A BRIL Adenosine receptor / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41 KDa

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Macromolecule #1: Adenosine receptor A2a,Soluble cytochrome b562

MacromoleculeName: Adenosine receptor A2a,Soluble cytochrome b562 / 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 aff. frugiperda 1 BOLD-2017 (butterflies/moths)
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

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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 / ZM-241,385

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Macromolecule #3: CHOLESTEROL

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

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #4: OLEIC ACID

MacromoleculeName: OLEIC ACID / type: ligand / ID: 4 / Number of copies: 15 / Formula: OLA
Molecular weightTheoretical: 282.461 Da
Chemical component information

ChemComp-OLA:
OLEIC ACID / Oleic acid

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Macromolecule #5: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate

MacromoleculeName: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / type: ligand / ID: 5 / Number of copies: 5 / Formula: OLC
Molecular weightTheoretical: 356.54 Da
Chemical component information

ChemComp-OLC:
(2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate

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Macromolecule #6: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate

MacromoleculeName: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / type: ligand / ID: 6 / Number of copies: 1 / Formula: OLB
Molecular weightTheoretical: 356.54 Da
Chemical component information

ChemComp-OLB:
(2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate

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Macromolecule #7: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 7 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 174 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration25 mg/mL
BufferpH: 4.7
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMilled microcrystals

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 1202 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystallography statisticsNumber intensities measured: 569407 / Number structure factors: 64974 / Fourier space coverage: 87.58 / R sym: 0.073 / R merge: 0.236 / Overall phase error: 30 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 0.87 Å / Shell - Shell ID: 1 / Shell - High resolution: 0.87 Å / Shell - Low resolution: 0.9 Å / Shell - Number structure factors: 2783 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 37.64 / Shell - Multiplicity: 2.1
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Merging software listSoftware - Name: AIMLESS
DetailsBinned by 2

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 12.54 / Target criteria: Maximum likelihood
Output model

PDB-8fyn:
MicroED structure of A2A from plasma milled lamellae

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