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

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Basic information

Entry
Database: PDB / ID: 8fyn
TitleMicroED structure of A2A from plasma milled lamellae
ComponentsAdenosine receptor A2a,Soluble cytochrome b562
KeywordsMEMBRANE PROTEIN / A2A Adenosine Receptor
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
CHOLESTEROL / OLEIC ACID / (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / Chem-ZMA / Soluble cytochrome b562 / Adenosine receptor A2a
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2 Å
AuthorsMartynowycz, M.W. / Shiriaeva, A. / Clabbers, M.T.B. / Nicolas, W.J. / Weaver, S.J. / Hattne, J. / Gonen, T.
Funding support United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Source and taxonomy / Category: entity_src_gen / Item: _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id
Revision 1.2Oct 23, 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

Downloads & links

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Assembly

Deposited unit
A: Adenosine receptor A2a,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,87127
Polymers49,9741
Non-polymers7,89626
Water3,135174
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)39.080, 177.510, 139.290
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Components on special symmetry positions
IDModelComponents
11A-2666-

HOH

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 49974.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADORA2A, ADORA2, cybC
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P29274, UniProt: P0ABE7

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Non-polymers , 7 types, 200 molecules

#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 / Comment: antagonist*YM
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical
ChemComp-OLA / OLEIC ACID


Mass: 282.461 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C18H34O2
#5: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C21H40O4
#6: Chemical ChemComp-OLB / (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate


Mass: 356.540 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H40O4
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 174 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: A2A BRIL Adenosine receptor / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.041 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 4.7
SpecimenConc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Milled microcrystals
Specimen supportGrid type: Quantifoil R2/2
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Data collection

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: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.5 sec. / Electron dose: 0.001 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1202 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 0.87→0.9 Å / Fourier space coverage: 37.64 % / Multiplicity: 2.1 / Num. of structure factors: 2783 / Phase residual: 30 °
EM diffraction statsFourier space coverage: 87.58 % / High resolution: 0.87 Å / Num. of intensities measured: 569407 / Num. of structure factors: 64974 / Phase error: 30 ° / Phase error rejection criteria: None / Rmerge: 0.236 / Rsym: 0.073
ReflectionBiso Wilson estimate: 12.54 Å2

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Processing

Software
NameVersionClassificationNB
phenix.refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: AIMLESS / Category: crystallography merging
Image processingDetails: Binned by 2
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 39.08 Å / B: 177.51 Å / C: 139.29 Å / Space group name: 20 / Space group num: 20
CTF correctionType: NONE
3D reconstructionResolution: 2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 12.54 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
RefinementResolution: 2→41.14 Å / SU ML: 0.2918 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 28.6157
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2792 1261 5.21 %
Rwork0.2202 22958 -
obs0.2233 24219 72.51 %
Solvent computationShrinkage radii: 0.6 Å / VDW probe radii: 0.9 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 23.1 Å2
Refinement stepCycle: LAST / Resolution: 2→41.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3012 0 436 174 3622
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00183501
ELECTRON CRYSTALLOGRAPHYf_angle_d0.6064675
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.1075527
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0028547
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d18.1672658
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.080.2306240.3186590ELECTRON CRYSTALLOGRAPHY16.83
2.08-2.180.3142670.28071136ELECTRON CRYSTALLOGRAPHY32.75
2.18-2.290.29841060.26762207ELECTRON CRYSTALLOGRAPHY63.6
2.29-2.430.29991660.2682803ELECTRON CRYSTALLOGRAPHY80.72
2.43-2.620.31661450.25262808ELECTRON CRYSTALLOGRAPHY80.35
2.62-2.890.32191900.24713134ELECTRON CRYSTALLOGRAPHY90.72
2.89-3.30.30371830.23493419ELECTRON CRYSTALLOGRAPHY96.18
3.3-4.160.23791790.18043398ELECTRON CRYSTALLOGRAPHY95.51
4.16-41.140.24982010.17543463ELECTRON CRYSTALLOGRAPHY93.33

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