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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 ION
  • Ligand: water
KeywordsA2A Adenosine Receptor / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of norepinephrine secretion / positive regulation of circadian sleep/wake cycle, sleep / positive regulation of acetylcholine secretion, neurotransmission / negative regulation of alpha-beta T cell activation / 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 ...regulation of norepinephrine secretion / positive regulation of circadian sleep/wake cycle, sleep / positive regulation of acetylcholine secretion, neurotransmission / negative regulation of alpha-beta T cell activation / 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 / inhibitory postsynaptic potential / type 5 metabotropic glutamate receptor binding / negative regulation of vascular permeability / positive regulation of glutamate secretion / synaptic transmission, cholinergic / response to caffeine / blood circulation / intermediate filament / eating behavior / alpha-actinin binding / presynaptic active zone / regulation of calcium ion transport / membrane depolarization / asymmetric synapse / axolemma / phagocytosis / prepulse inhibition / cellular defense response / presynaptic modulation of chemical synaptic transmission / response to amphetamine / positive regulation of synaptic transmission, glutamatergic / neuron projection morphogenesis / regulation of mitochondrial membrane potential / central nervous system development / positive regulation of apoptotic signaling pathway / positive regulation of long-term synaptic potentiation / excitatory postsynaptic potential / positive regulation of synaptic transmission, GABAergic / synaptic transmission, glutamatergic / positive regulation of protein secretion / locomotory behavior / astrocyte activation / apoptotic signaling pathway / electron transport chain / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / negative regulation of inflammatory response / adenylate cyclase-activating G protein-coupled receptor signaling pathway / blood coagulation / vasodilation / 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 / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / calmodulin binding / inflammatory response / iron ion binding / response to xenobiotic stimulus / negative regulation of cell population proliferation / neuronal cell body / apoptotic process / lipid binding / heme 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 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.49 Å/pix.
x 145 pix.
= 39.08 Å		0.49 Å/pix.
x 221 pix.
= 175.86 Å		0.48 Å/pix.
x 118 pix.
= 139.29 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.4885 Å / Y: 0.4885 Å / Z: 0.48365 Å
Density

Histogram

Histogram (log scale)
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 ION
  • Ligand: water

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Supramolecule #1: A2A BRIL Adenosine receptor

SupramoleculeName: A2A BRIL Adenosine receptor / type: complex / ID: 1 / 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

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: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
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

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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

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMilled microcrystals

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsBinned by 2
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Merging software listSoftware - Name: AIMLESS
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

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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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