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

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Entry
Database: PDB / ID: 7rm5
TitleMicroED structure of the human adenosine receptor at 2.8A
ComponentsAdenosine receptor A2a/Soluble cytochrome b562 chimera
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
CHOLESTEROL / Chem-ZMA / Soluble cytochrome b562 / Adenosine receptor A2a
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.79 Å
AuthorsMartynowycz, M.W. / Shiriaeva, A. / Ge, X. / Hattne, J. / Nannenga, B.L. / Cherezov, V. / Gonen, T.
Funding support United States, 4items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 15, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Oct 26, 2022Group: Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen ...pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details ..._pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.oper_expression
Revision 1.3Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)51,8817
Polymers49,9741
Non-polymers1,9076
Water1086
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)40.005, 180.540, 139.670
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

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Components

#1: Protein Adenosine receptor A2a/Soluble cytochrome b562 chimera / 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. exp.) Escherichia coli (E. coli)
Gene: ADORA2A, ADORA2, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29274, UniProt: P0ABE7
#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.: 4 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical ChemComp-NA / SODIUM ION


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


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: Adenosine receptor A2a/Soluble cytochrome b562 chimera
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.05872 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Spodoptera frugiperda (fall armyworm)7108
21Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 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
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 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): 83 K / Temperature (min): 77 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 134 / Num. of grids imaged: 1
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1900 mm / Tilt angle list: -40, +40, 0.6
EM diffraction shellResolution: 37.91→2.79 Å / Fourier space coverage: 77 % / Multiplicity: 3.7 / Num. of structure factors: 10071 / Phase residual: 30 °
EM diffraction statsFourier space coverage: 77 % / High resolution: 2.79 Å / Num. of intensities measured: 37130 / Num. of structure factors: 10071 / Phase error: 30 ° / Phase error rejection criteria: none / Rmerge: 0.299
DiffractionSerial crystal experiment: N
DetectorDetector: CMOS / Date: Sep 11, 2020

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Processing

Software
NameClassification
DIALSdata reduction
PHASERphasing
PHENIXrefinement
EM software
IDNameCategory
1EPUimage acquisition
6Cootmodel fitting
8PHENIXmodel refinement
9PHASERmolecular replacement
11DIALSsymmetry determination
13PHENIX3D reconstruction
Image processingDetails: CetaD binned by 2
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 40 Å / B: 180.5 Å / C: 139.7 Å / Space group name: C2221 / Space group num: 20
CTF correctionType: NONE
3D reconstructionResolution: 2.79 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 43 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum likelihood
Atomic model buildingPDB-ID: 4EIY

4eiy


Pdb chain-ID: A / Accession code: 4EIY / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4EIY

4eiy


Resolution: 2.79→37.91 Å / SU B: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.3 / Phase error: 0.3063
RfactorNum. reflection% reflectionSelection details
Rfree0.2881 525 5.21 %Copied from 4EIY
Rwork0.2482 ---
obs0.2503 10035 77.12 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 43 Å2
LS refinement shellResolution: 2.79→3.07 Å
RfactorNum. reflection% reflection
Rfree0.3648 124 5 %
Rwork0.32 2268 -
obs--75 %

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